Screening methods for identifying viral proteins with interferon antagonizing functions and potential antiviral agents

ABSTRACT

The present invention relates, in general, to a screening method for identifying novel viral proteins with interferon antagonizing function using a transfection-based assay, and the use of such proteins in isolating various types of attenuated viruses for the development of vaccine and pharmaceutical formulations. The invention also relates to the use of viral interferon antagonists in screening assays to identify potential anti-viral agents. The invention further relates to protocols utilizing interferon antagonists, e.g., NS1, to enhance gene therapy or DNA vaccination based on their ability to increase gene expression.

1. INTRODUCTION

The present invention relates, in general, to a screening method foridentifying novel viral proteins with interferon antagonizing function,and the use of such proteins in isolating various types of attenuatedviruses for the development of vaccine and pharmaceutical formulations.The invention also relates to the use of viral interferon antagonists inscreening assays to identify potential anti-viral agents. The inventionfurther relates to protocols utilizing interferon antagonists, e.g.,NS1, to enhance gene therapy or DNA vaccination based on their abilityto increase gene expression.

2. BACKGROUND OF THE INVENTION

One important component of the host antiviral response is the type I IFNsystem. Type I IFN is synthesized in response to viral infection. Doublestranded RNA (dsRNA) or viral infection activate latent transcriptionfactors, including IRF-3 and NF-_(k)B, resulting in transcriptionalup-regulation of type I IFN, IFN-α, and IFN-β genes. Secreted type IIFNs signal through a common receptor, activating the JAK/STAT signalingpathway. This signaling stimulates transcription of IFN-sensitive genes,including a number of that encode antiviral proteins, and leads to theinduction of an antiviral state. Among the antiviral proteins induced inresponse to type I IFN are dsRNA-dependent protein kinase R (PKR).2′,5′-oligoadenylate synthetase (OSA), and the Mx proteins (Clemens etal., 1997 Interferon Cytokine Res. 17:503-524; Floyd-Smith et al., 1981Science 212:1030-1032; Haller et al., 1998 Rev. Sci Tech 17:220-230;Stark et al., Annu Rev. Biochem 67:227-264).

Many viruses have evolved mechanisms to subvert the host IFN response.For example, the herpes simplex virus counteracts the PKR-mediatedphosphorylation of translation initiation factor cIF-2α, preventing theestablishment of an IFN-induced block in protein synthesis(Garcia-Sastre et al. 1998 Virology 252(2):324-30). In thenegative-strand RNA viruses, several different anti-IFN mechanisms havebeen identified (Garcia-Sastre et al., 1998 Virology 252:324-330).

Citation of a reference in this section or any section of thisapplication shall not be construed as an admission that such referenceis prior art to the present invention.

3. SUMMARY OF THE INVENTION

The invention relates to screening methods for viral proteins withinterferon antagonizing function based on transfection-based assaysusing various types of negative strand RNA viruses. The identifiedinterferon antagonists can be used for several applications. Theinvention relates to attenuated viruses having an impaired ability toantagonize the cellular interferon (IFN) response, and the use of suchattenuated viruses in vaccine and pharmaceutical formulations. Further,the present invention relates to viruses which have been mutated toimpair the virus's ability to antagonize cellular interferon responses,impaired viruses or viruses with impaired interferon antagonistactivity. The present invention also relates to growth substrates whichsupport the growth of viruses, both naturally occurring and mutagenized,which have an impaired ability to antagonize the cellular interferonresponse, for diagnostic or therapeutic purposes.

The present invention relates to transfection-based assays to identifyviral proteins with interferon-antagonizing activities. Once such viralproteins have been identified, genes encoding these proteins can betargeted to create attenuated viruses for the development of vaccines.Further, the viral proteins identified to have interferon-antagonizingactivities can be used to support the growth of viruses with impairedabilities to antagonize cellular interferon responses for diagnostic,therapeutic or research protocols.

In a preferred embodiment, the present invention relates to screeningassays to identify potential antiviral agents which inhibit the abilityof the virus to antagonize cellular interferon responses. Thus, theidentified viral proteins which antagonize interferon responses willalso have utility in screening for and developing novel antiviralagents.

The present invention also relates to the substrates designed for theisolation, identification and growth of viruses for vaccine purposes aswell as diagnostic and research purposes. In particular,interferon-deficient substrates for efficiently growing influenza virusmutants are described. In accordance with the present invention, aninterferon-deficient substrate is one that is defective in its abilityto produce or respond to interferon. The substrate of the presentinvention may be used for the growth of any number of viruses which mayrequire interferon-deficient growth environment.

Furthermore, cell lines expressing viral proteins withinterferon-antagonizing properties are encompassed by the presentinvention. These proteins include, for example, NS1 and other analogousproteins originating from various types of viruses. Such viruses mayinclude, but are not limited to paramyxoviruses (Sendai virus,parainfluenza virus, mumps, Newcastle disease virus), morbilliviruses(measles virus, canine distemper virus and rinderpest virus);pneumoviruses (respiratory syncytial virus and bovine respiratoryvirus); rhabdoviruses (vesicular stomatitis virus and lyssavirus); RNAviruses, including hepatitis C virus and retroviruses, and DNA viruses,including vaccinia, adenoviruses, hepadna viruses, herpes viruses andpoxviruses.

Any number of viruses may be used in accordance with the presentinvention, including DNA viruses, e.g., vaccinia, adenoviruses, hepadnaviruses, herpes viruses, poxviruses, and parvoviruses; and RNA viruses,including hepatitis C3 virus, retrovirus, and segmented andnon-segmented RNA viruses. The viruses can have segmented ornon-segmented genomes and can be selected from naturally occurringstrains, variants or mutants; mutagenized viruses (e.g., by exposure toUV irradiation, mutagens, and/or passaging); reassortants (for viruseswith segmented genomes); and/or genetically engineered viruses. Forexample, the mutant viruses can be generated by natural variation,exposure to UV irradiation, exposure to chemical mutagens, by passagingin non-permissive hosts, by reassortment (i.e., by coinfection of anattenuated segmented virus with another strain having the desiredantigens), and/or by genetic engineering (e.g., using “reversegenetics”). The viruses selected for use in the invention have defectiveIFN antagonist activity and are attenuated; i.e., they are infectiousand can replicate in vivo, but only generate low titers resulting insubclinical levels of infection that are non-pathogenic. Such attenuatedviruses are ideal candidates for live vaccines.

The invention is based, in part, on a number of discoveries andobservations made by the Applicants when working with influenza virusmutants. However, the principles can be analogously applied andextrapolated to other segmented and non-segmented negative strand RNAviruses including, but not limited to paramyxoviruses (Sendai virus,parainfluenza virus, mumps, Newcastle disease virus), morbilliviruses(measles virus, canine distemper virus and rinderpest virus);pneumoviruses (respiratory syncytial virus and bovine respiratoryvirus); and rhabdoviruses (vesicular stomatitis virus andlyssavirus),and vaccinia, adenoviruses, hepadna viruses, herpes virusesand poxviruses.

First, the IFN response is important for containing viral infection invivo. The Applicants found that growth of wild-type influenza virusA/WSN/33 in IFN-deficient mice (STAT1−/−mice) resulted in pan-organinfection; i.e., viral infection was not confined to the lungs as it isin wild-type mice which generate an IFN response (Garcia-Sastre, et al.,1998, J. Virol. 72:8550, which is incorporated by reference herein inits entirety). Second, the Applicants established that NS1 of influenzavirus functions as an IFN antagonist.

The invention also relates to the use of the attenuated virus of theinvention in vaccines and pharmaceutical preparations for humans oranimals. In particular, the attenuated viruses can be used as vaccinesagainst a broad range of viruses and/or antigens, including but notlimited to antigens of strain variants, different viruses or otherinfectious pathogens (e.g., bacteria, parasites, fungi), or tumorspecific antigens. In another embodiment, the attenuated viruses, whichinhibit viral replication and tumor formation, can be used for theprophylaxis or treatment of infection (viral or nonviral pathogens) ortumor formation or treatment of diseases for which IFN is of therapeuticbenefit. Many methods may be used to introduce the live attenuated virusformulations to a human or animal subject to induce an immune orappropriate cytokine response. These include, but are not limited to,intranasal, intratrachial, oral, intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous routes. In a preferredembodiment, the attenuated viruses of the present invention areformulated for delivery intranasally.

The specifications of application serial Nos. WO99/64571; WO99/64068;and WO99/64570, are each incorporated herein by reference in theirentireties.

3.1. DEFINITIONS

“Isolated” or “purified” when used herein to describe a protein orbiologically active portion thereof (i.e., a polypeptide, peptide oramino acid fragment), refers to a protein or biologically active portionthereof substantially free of cellular material or other contaminatingproteins from the cell or tissue source from which the protein isderived, or substantially free of chemical precursors or other chemicalswhen chemically synthesized. A protein or biologically active portionthereof (i.e., a polypeptide, peptide or amino acid fragment) that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”).

In certain embodiments of the invention, a “prophylactically effectiveamount” is the amount of a composition of the invention that reduces theincidence of cancer, viral infection, or microbial infection, in ananimal. Preferably, the incidence of cancer, viral infection, ormicrobial infection in an animal is reduced by at least 2.5%, at least5%, at least 10%, at least 15%, at least 25%, at least 35%, at least45%, at least 50%, at least 75%, at least 85%, by at least 90%, at least95%, or at least 99% in an animal administered a composition of theinvention relative to an animal or group of animals (e.g., two, three,five, ten or more animals) not administered a composition of theinvention.

In certain embodiments of the invention, a “therapeutically effectiveamount” is the amount of a composition of the invention that reduces theseverity, the duration and/or the symptoms associated with cancer, viralinfection, or microbial infection, in an animal. In certain otherembodiments of the invention, a “therapeutically effective amount” isthe amount of a composition of the invention that results in a reductionin viral titer or microbial titer by at least 2.5%, at least 5%, atleast 10%, at least 15%, at least 25%, at least 35%, at least 45%, atleast 50%, at least 75%, at least 85%, by at least 90%, at least 95%, orat least 99% in an animal administered a composition of the inventionrelative to the viral titer or microbial titer in an animal or group ofanimals (e.g., two, three, five, ten or more animals) not administered acomposition of the invention. In certain other embodiments, a“therapeutically effective amount” is the amount of a composition of theinvention that results in a reduction of the growth or spread of cancerby at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%,at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, byat least 90%, at least 95%, or at least 99% in an animal administered acomposition of the invention relative to the growth or spread of cancerin an animal or group of animals (e.g., two, three, five, ten or moreanimals) not administered a composition of the invention.

4. DESCRIPTION OF THE FIGURES

FIG. 1. System to identify viral encoded interferon antagonists. Cellsare transfected with plasmids encoding known or potentialinterferon-antagonists. Sixteen hours later, the cells are infected withan interferon-sensitive virus, such as delNS1 virus. Viral growth isthen monitored. Effective interferon-antagonists will block interferoninduction and subsequent activation of antiviral pathways. The result isenhanced viral growth.

FIG. 2. Method to enhance growth of Interferon-sensitive viruses. Cellswill be transfected with a plasmid encoding an interferon-antagonist andsubsequently infected with the interferon-sensitive virus. Inhibition ofthe interferon response by the interferon antagonist will enhance virusgrowth.

FIG. 3. Screening assay to identify inhibitors ofinterferon-antagonists. Compounds will be screened for their ability toinhibit interferon antagonists. Cells containing a reporter plasmid withan interferon-stimulated response element driven GFP (ISRE-GFP) andexpressing an interferon antagonist will be infected with a virus withimpaired interferon antagonist activity (e.g., delNS1). These infectedcells will also be treated with different test compounds.

FIG. 3A. In the presence of a compound (compound A) which does notinhibit the interferon antagonist, interferon response is not induced.Therefore, GFP signal is not detected and growth of the virus withimpaired interferon antagonist activity is detected.

FIG. 3B. In the presence of a compound (compound B) which inhibits theinterferon antagonist, interferon is produced, GFP expression isdetected and growth of the virus with impaired interferon antagonistactivity is not detected.

FIG. 4. Stimulation of luciferase expression from pGL2-Control byco-expression with a viral interferon antagonist. Transfection of aninterferon antagonist can enhance expression of other genes. The abilityto enhance expression of transfected genes may be useful when maximalgene expression is desired.

Interferon antagonists may enhance expression in vivo from gene therapyvectors.

FIG. 5. Growth of the influenza delNS1 virus is complemented bytransient transfection of an influenza A NS1 protein or an HSV ICP34.5expression plasmid. MDCK cells were transfected with 4 μg of emptyexpression plasmid (pCAGGS), pCAGGS-PR8 NS1 SAM, or pCAGGS-134.5.Twenty-four hours later, the cells were infected with influenza delNS1virus (moi=0.001). Forty-eight hours posttransfection, viral titers weredetermined by plaque assay. The results are the average of twoindependent experiments.

FIG. 6. Growth of the influenza delNS1 virus is complemented by theEbola virus VP35 protein. MDCK cells were transfected with 4 μg of emptyexpression plasmid (pcDNA3), NS1 expression plasmid, or Ebola virus VP35expression plasmid. Twenty-four hours later, the cells were infectedwith influenza delNS1 virus (moi=0.001). Viral titers were determined byplaque assay at the indicated times.

FIG. 7. Expression of Ebola virus VP35 protein inhibits dsRNA- orvirus-mediated induction of an ISRE.

FIG. 7A. Fold induction of an ISRE promoter-CAT reporter gene in thepresence of empty vector, NS1 expression plasmid, or VP35 expressionplasmid. The CAT activities were normalized to the correspondingluciferase activities to determine fold induction.

FIG. 7B. Western blot showing NS1, VP35, and Ebola virus NP expression.293 cells were transfected with 4 μg of the indicated plasmids.Forty-eight hours later, cell lysates were prepared and Western blotswere performed by using the indicated antiserum.

FIG. 8. The VP35 protein of Ebola virus inhibits induction of the IFN-•promoter.

FIG. 8A. Inhibition of induction of the mouse IFN-β promoter. 293 cellswere transfected with 4 μg of the indicated expression plasmid plus 0.3μg each of the reporter plasmids pIFN-β-CAT and pGL2-Control.Twenty-four hours posttransfection, the cells were mock-transfected ortransfected with 40 μg of polyI:polyC.

FIG. 8B. Northern blot showing VP35-mediated inhibition of endogenousIFN-induction. 293 cells were transfected with either empty vector orVP35 expression plasmid. Twenty-four hours later, the cells weremock-infected or infected with influenza delNS1 virus (delNS1) or Sendaivirus (SeV) (moi=1). Total RNA was prepared from cells at ten or twentyhours posttransfection. Mock-transfected cell RNA was prepared at thesame time as the twenty hour postinfection samples. Northern blots wereperformed to detect IFN-β or β-actin mRNAs.

Note that less total RNA was obtained when cells, including themock-infected cells, were lysed at the twenty hour postinfection timepoint.

FIG. 9. The Ebola virus VP35 protein inhibits type I IFN induction whencoexpressed with Ebola virus NP. Fold induction of the IFN-inducibleISRE-driven reporter in the presence of empty vector, VP35, NP, or VP35plus NP. 293 cells were transfected with a total of 4 μg of expressionplasmid, including 2 μg of a plasmid encoding an individual protein and2 μg of a second plasmid (either empty vector or a second expressionplasmid) plus 0.3 μg each of the reporter plasmids pHISG-54-CAT andpGL2-Control. Twenty-four hours posttransfection, the cells weremock-treated or treated with the indicated IFN inducer. Twenty-fourhours postinduction, CAT and luciferase assays were performed. The CATactivities were normalized to the corresponding luciferase activities todetermine fold induction.

5. DETAILED DESCRIPTION OF INVENTION

The invention relates to screening assays to identify viral proteinswith interferon antagonizing function. The present invention relates toidentifying viral proteins that have the ability to complementreplication of an attenuated virus with impaired ability to antagonizecellular interferon responses. The present invention also relates toscreening assays to identify anti-viral agents which inhibit interferonantagonist activity and inhibit viral replication.

The screening assays of the invention are based, in part, on Applicants'discovery that viral proteins such as influenza NS1, ebola virus VP35and respiratory syncytial virus NS2 function as an IFN antagonists, inthat these proteins inhibit the IFN mediated response of virus-infectedcells. However, the principles can be analogously applied andextrapolated to other viruses, including other segmented andnon-segmented RNA viruses, such viruses may include, but are not limitedto paramyxoviruses (Sendai virus, parainfluenza virus, mumps, Newcastledisease virus), morbillivirus (measles virus, canine distemper virus andrinderpest virus); pneumovirus (respiratory syncytial virus and bovinerespiratory virus); rhabdovirus (vesicular stomatitis virus andlyssavirus); RNA viruses, including Hepatitis C virus and retroviruses,lentiviruses, including human immunodeficiency virus (HIV), and DNAviruses, including vaccinia, adenoviruses, adeno-associated virus,hepadna viruses, herpes viruses and poxviruses.

The present invention relates to in vitro and cell based assays toidentify viral proteins with interferon antagonizing function. In apreferred embodiment, the present invention relates totransfection-based assays to identify viral proteins withinterferon-antagonizing activities. In one embodiment, thetransfection-based assays of the invention encompass expressing theputative interferon antagonist in a cell infected with a virus withimpaired ability to antagonize cellular interferon functions. Interferonantagonist activity may be determined by the ability of the viralprotein to complement replication of the impaired virus. The ability ofan interferon antagonist to complement replication of an impaired virus,i.e., a virus in which the interferon antagonist activity is mutated orreduced, may be determined in a cell based or animal based assay. Ineither assay system, the ability of the interferon antagonist tocomplement the impaired virus is determined by an increase or anenhancement in viral replication of viral load.

In accordance with the screening assays of the present invention,numerous in vitro and cell based assays may be used to identify viralproteins with interferon antagonist activity. Interferon antagonistactivities may be determined by the ability of a viral protein toinhibit or reduce any known interferon based activity, includingregulation of interferon expression, regulation of interferon regulatedpromoter elements and genes, regulation of signal transduction pathways,such as the phosphorylation of Janus Kinases (JAKS) and signaltransduction activator of transcription (STATS).

The present invention relates to screening methods to identify potentialantiviral agents that target interferon antagonists. The presentinvention relates to screening assays based on identifying agents whichinhibit interferon antagonizing activity. The antiviral screening assaysof the invention encompass in vitro, in vivo and animal models foridentifying antiviral agents that target interferon antagonists.

The ability of an agent or compound to target or modulate a viralinterferon antagonist may be determined by measuring the ability of saidagent or compound to modulate or regulate, either directly orindirectly, the viral protein's inhibition of cellular interferonresponses. The invention encompasses screening for an agent or compoundwith the ability to target or modulate viral interferon antagonistactivities, including the ability of a viral protein to inhibit orreduce any known interferon based activity, including regulation ofinterferon expression, regulation of interferon regulated promoterelements and genes, regulation of signal transduction pathways, such asthe phosphorylation of Janus Kinases (JAKS) and signal transductionactivator of transcription (STATS).

The present invention also provides cell and animal based models for theidentification of an agent or compound to target or modulate a viralinterferon antagonist and inhibit or reduce viral replication. The celland animal based model of the invention comprising measuring the abilityof a test agent or compound to inhibit the complementation of a viruswith impaired interferon antagonist activity by a viral interferonantagonist.

In such an assay system, the interferon antagonist may be provided tothe virus with impaired interferon antagonistic in trans or in cis. Aninterferon antagonist may be provided to the cell or animal system intrans by providing the nucleic acids encoding said interferon antagonistor the interferon antagonist polypeptide using standard techniques knownto those of skill in the art. An interferon antagonist may be providedin cis by constructing a chimeric virus comprising a nucleic acidencoding said interferon antagonist and nucleic acids encoding the viruswith impaired interferon antagonist activity.

In accordance with the present invention, the identified viralinterferon antagonists can be used for several applications. Viralinterferon antagonists can be used as targets for mutagenesis aimed atcreating viruses with impaired interferon antagonist activity andattenuated phenotypes. Viral interferon antagonists can be used toenhance growth of viruses that display restricted growth on interferonproducing substrates. Such growth substrates may allow the isolation andcharacterization of interferon sensitive viruses and may increase viraltiters obtained in tissue culture. Viral interferon antagonists may beused to enhance translation of co-expressed genes. This capability maybe useful in maximizing expression of transfected genes. Viralinterferon antagonists may be used to facilitate gene therapy or DNAvaccination by increasing and/or prolonging gene expression in thepresence of interferon.

The present invention also encompasses pharmaceutical compositionscomprising antiviral agents which inhibit viral interferon antagonistactivity and methods of administering such pharmaceutical compositionsfor the treatment and prevention of viral replication.

5.1. SCREENING ASSAYS FOR IDENTIFYING VIRAL PROTEINS HAVING INTERFERONANTAGONIST ACTIVITY

The present invention relates to screening methods to identify viralproteins with interferon antagonizing function. The screening assays ofthe invention encompass in vitro and in vivo approaches to assay for theability of a viral protein to antagonize cellular interferon responses.In accordance with the present invention, interferon antagonistactivities may be determined by the ability of a viral protein toinhibit or reduce any known interferon based activity, as compared tothe absence of the viral protein. Interferon based activities which maybe assayed include, but are not limited to, regulation of interferonregulated promoter elements and genes, regulation of reporter genes,increase in translation of proteins, and regulation of signaltransduction pathways, such as the phosphorylation of JAKS and STATS.

In a preferred embodiment, the present invention relates to acomplementation assay. A complementation assay allows for the screeningof a viral protein to determine if it possesses an interferon antagonistactivity thereby complementing the growth of a virus lacking saidactivity.

A complementation assay consists of using an appropriate cell line thatis susceptible to the virus from which the virus with impairedinterferon antagonist activity is derived. The assay comprisesdetermining the ability of a viral protein to complement the interferonantagonist function and allow the virus to replicate and grow. Any viralprotein may be assayed for interferon antagonist activity.

Increased viral replication in the presence of the viral protein orpeptide, compared to the absence of said protein or peptide, indicatesthe viral protein or peptide has interferon antagonist activity.

In accordance with the present invention, the screening assays toidentify viral interferon antagonists may be performed in anyappropriate in vitro assay system, cell extract or cell, includingprimary cells and cell lines.

The viral protein to be tested for interferon antagonist activity can beobtained from any virus. These proteins include, for example, NS1 andother analogous proteins originating from various types of viruses. Suchviruses may include, but are not limited to paramyxoviruses (Sendaivirus, parainfluenza virus, mumps, Newcastle disease virus),morbilliviruses (measles virus, canine distemper virus and rinderpestvirus); pneumoviruses (respiratory syncytial virus and bovinerespiratory virus); rhabdoviruses (vesicular stomatitis virus andlyssavirus); RNA viruses, including hepatitis C virus and retroviruses,and DNA viruses, including vaccinia, adenoviruses, hepadna viruses,herpes viruses and poxviruses.

In accordance with the present invention, the viral proteins to betested for interferon antagonist activity may be provided to the assaysystem to be used as an isolated protein or fragment thereof or inanother embodiment, the nucleic acids encoding the viral protein or aportion thereof may be provided to the assay system.

Viral proteins to be tested for interferon antagonist activity may beisolated or purified from a virus or viral extract using standardtechniques known to those of skill in the art. In another embodiment theviral protein to be tested may be expressed recombinantly using standardtechniques known to those of skill in the art. Nucleic acids encodingviral proteins to be tested for interferon antagonist activity may besupplied using standard techniques known to those of skill in the art.Nucleic acids encoding viral proteins to be tested should be operativelylinked to the appropriate regulatory elements to allow for theirexpression. Such nucleic acids may be supplied by way of plasmid, viralvector, bacteriophage etc. and may be operatively linked to regulatoryelements selected from viral promoter elements, inducible promoters,constitutive promoters etc.

The ability of a viral protein to antagonize interferon responses isdetermined by the ability of the viral protein to reduce or inhibit theinterferon response being assayed, as compared to the absence of theviral protein.

5.1.1. Complementation Assays

In a preferred embodiment, the present invention encompasses screeningassays to identify viral interferon antagonists based on the ability ofthe viral protein to complement the growth and replication of a viruswith impaired interferon antagonist activity, as compared to the absenceof the viral protein.

In accordance with the embodiment of the invention, the viral protein tobe tested for its ability to complement the growth and replication of avirus with impaired interferon antagonist activity is provided to theimpaired virus in trans. The viral protein or nucleic acids encoding theviral protein, and the impaired virus is provided as a packaged virionor the nucleic acids encoding the impaired virus are provided to thecell. In such an embodiment, the cell may be engineered to express thecomponents of the assay using standard techniques available to thoseskilled in the art. In such an embodiment, the growth and replication ofthe virus is compared in the presence and the absence of the viralprotein.

In a preferred embodiment the viral protein to be tested for its abilityto complement the growth and replication of a virus with impairedinterferon antagonist activity is provided to the impaired virus in cis.In such an embodiment, a chimeric virus is engineered so that thechimeric virus expresses the viral protein to be tested. In such anembodiment, the growth and replication of the impaired virus is comparedto that of the chimeric virus.

In a preferred embodiment the virus with impaired interferon antagonistactivity is influenza delNS1, however, any virus with impairedinterferon antagonist activity can be used in accordance with theinvention.

The present invention encompasses chimeric viruses wherein the virus hasa defect such that it is impaired in its interferon antagonist activityand said defect is complemented by the presence of a heterologousinterferon antagonist. The chimeric virus can be made using any RNAvirus including negative strand RNA virus that are either segmented ornon-segmented. In a preferred embodiment the chimeric virus isengineered using an influenza virus.

“Reverse genetics” techniques are used to construct recombinant and/orchimeric influenza virus templates engineered to direct the expressionof heterologous gene products. When combined with purified viralRNA-directed RNA polymerase, these virus templates are infectious,replicate in hosts, and their heterologous gene is expressed andpackaged by the resulting recombinant influenza viruses (For adescription of the reverse genetics approach see Palese et al., U.S.Pat. No. 5,166,057 and Palese, WO93/21306, each of which is incorporatedby reference herein in its entirety). The expression products and/orchimeric virions obtained can be used in vaccine formulations, and thestrain variability of the influenza virus permits construction of a vastrepertoire of vaccine formulations and obviates the problem of hostresistance.

The use of reverse genetics to genetically engineer influenza viruses,including attenuated influenza viruses, and methods for theirproduction, are described in Palese et al.. (U.S. Pat. No. 5,166,057)and Palese (WO93/21306). Such reverse genetics techniques can beutilized to engineer a mutation, including but not limited to aninsertion, deletion, or substitution of an amino acid residue(s), anantigen(s), or an epitope(s) into a coding region of the viral genome sothat altered or chimeric viral proteins are expressed by the engineeredvirus. Alternatively, the virus can be engineered to express theinterferon antagonist as an independent polypeptide.

The reverse genetics technique involves the preparation of syntheticrecombinant viral RNAs that contain the non-coding regions of thenegative strand virus which are essential for the recognition of viralRNA by viral polymerases and for the packaging into mature virions. Therecombinant RNAs are synthesized from a recombinant DNA template andreconstituted in vitro with purified viral polymerase and nucleoproteincomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells.

Preferably, the viral polymerase proteins are present during in vitrotranscription of the synthetic RNAs prior to transfection. The syntheticrecombinant RNPs can be rescued into infectious virus particles. Theforegoing techniques are described in Palese et al., U.S. Pat. No.5,166,057, and in Enami and Palese, 1991, J. Virol. 65:2711-2713, eachof which is incorporated by reference herein in its entirety.

Such reverse genetics techniques can be used to insert an interferonantagonist into an influenza virus protein so that a chimeric protein isexpressed by the virus. Any of the influenza viral proteins may beengineered in this way.

Alternatively, viral genes can be engineered to encode a viral proteinand the interferon antagonist as independent polypeptides. To this end,reverse genetics can advantageously be used to engineer a bicistronicRNA segment as described in U.S. Pat. No. 5,166,057, which isincorporated by reference in its entirety herein; i.e., so that theengineered viral RNA species directs the production of both the viralprotein and the interferon antagonist as independent polypeptides.

Attenuated strains of influenza may be used as the “parental” strain togenerate the influenza recombinants. Alternatively, reverse genetics canbe used to engineer both the attenuation characteristics as well as theinterferon antagonist into the recombinant influenza viruses of theinvention.

5.1.2 Interferon Activities to be Assayed

The screening methods of the invention also encompass identifying thoseviral proteins which antagonize IFN responses. In accordance with thescreening methods of the invention, induction of IFN responses may bemeasured by assaying levels of IFN expression or expression of targetgenes or reporter genes induced by IFN following transfection with theviral protein or activation of transactivators involved in the IFNexpression and/or the IFN response. Interferon antagonist activity canalso be determined by monitoring gene expression. This would includeendogenously expressed genes that are up regulated in response tointerferon or increased expression of a reporter gene linked to aninterferon responsive element (FIGS. 1 and 2).

In yet another embodiment of the selection systems of the invention,induction of IFN responses may be determined by measuring thephosphorylated state of components of the IFN pathway followingtransfection with the test viral protein, e.g., IRF-3, which isphosphorylated in response to double-stranded RNA. In response to type IIFN, Jak1 kinase and TyK2 kinase, subunits of the IFN receptor, STAT1,and STAT2 are rapidly tyrosine phosphorylated. Thus, in order todetermine whether the viral protein induces IFN responses, cells, suchas 293 cells, are transfected with the test viral protein and followingtransfection, the cells are lysed. IFN pathway components, such as Jak1kinase or TyK2 kinase, are immunoprecipitated from the infected celllysates, using specific polyclonal sera or antibodies, and the tyrosinephosphorylated state of the kinase is determined by immunoblot assayswith an anti-phosphotyrosine antibody (e.g., see Krishnan et al. 1997,Eur. J. Biochem. 247:298-305). An enhanced phosphorylated state of anyof the components of the IFN pathway following transfection with theviral protein would indicate induction of IFN responses by the viralprotein.

In yet another embodiment, the screening systems of the inventionencompass measuring the ability to bind specific DNA sequences or thetranslocation of transcription factors induced in response totransfection of a viral protein, e.g., IRF3, STAT1, STAT2, etc. Inparticular, STAT1 and STAT2 are phosphorylated and translocated from thecytoplasm to the nucleus in response to type I IFN. The ability to bindspecific DNA sequences or the translocation of transcription factors canbe measured by techniques known to those of skill in the art, e.g.,electromobility gel shift assays, cell staining, etc.

In yet another embodiment of the screening systems of the invention,induction of IFN responses may be determined by measuring IFN-dependenttranscriptional activation following transfection with the test viralprotein. In this embodiment, the expression of genes known to be inducedby IFN, e.g., Mx, PKR, 2-5-oligoadenylatesynthetase, majorhistocompatibility complex (MHC) class I, etc., can be analyzed bytechniques known to those of skill in the art (e.g., northern blots,western blots, PCR, etc.).

Alternatively, test cells such as human embryonic kidney cells or humanosteogenic sarcoma cells, are engineered to transiently orconstitutively express reporter genes such as luciferase reporter geneor chloramphenicol transferase (CAT) reporter gene under the control ofan interferon stimulated response element, such as the IFN-stimulatedpromoter of the ISG-54K gene (Bluyssen et al., 1994, Eur. J. Biochem.220:395-402). Cells are transfected with the viral protein and the levelof expression of the reporter gene compared to that in untransfectedcells or cells transfected with a plasmid lacking a test protein, oralternatively containing a viral protein known not to have interferonantagonist activity. An increase in the level of expression of thereporter gene following transfection with the viral protein wouldindicate that the viral protein is inducing an IFN response.

In a preferred embodiment the virus with impaired interferon antagonistactivity is the influenza A virus mutant delNS1 and the test protein canbe any viral protein. Interferon antagonist activity can be monitored byany of the methods described above including but not limited to theability of the viral protein to enhance viral replication; the abilityof the viral protein to enhance interferon regulated gene expression; orthe ability of the viral protein to enhance signal transduction inpathways induced by interferon activation.

In yet another preferred embodiment the virus is a chimeric mutant viruscomprised of a heterologous viral protein of interest and a mutationthat impairs the native interferon antagonist activity. Interferonantagonist activity can be monitored by any of the methods describedabove including but not limited to the ability of the viral protein toenhance viral replication; the ability of the viral protein to enhanceinterferon regulated gene expression; or the ability of the viralprotein to enhance signal transduction in pathways induced by interferonactivation.

5.1.3. In vivo Screening Assays for Identifying Viral Proteins havingInterferon Antagonist Activity

The screening assay can be performed in any appropriate animal model. Anappropriate animal model would be one that is susceptible to infectionwith the virus from which the virus with impaired interferon antagonistactivity is derived. The animal model may be any animal, preferably theanimal is a mouse, rat, rabbit or avian.

The complement assays of the present invention as described in Section5.1.1 may be applied to in vivo screening assays. The viral protein tobe tested could be administered to the animal in trans to the impairedvirus or in cis, such as a chimeric virus. If the viral protein to betested is to be provided in trans, the nucleic acid encoding the viralprotein to be tested in the form of a plasmid, or viral vector. Theviral protein to be tested could be provided to the animal model as aprotein or peptide.

In addition to the introduction of the viral protein to be tested, theanimal would also be infected with a virus with impaired interferonantagonist activity. In a preferred embodiment of the invention, thevirus with impaired interferon antagonist activity is influenza A virusdelNS1.

The test animal can be monitored for viral titer, or gene expression ofgenes endogenously regulated by interferon or an exogenous gene underthe control of an interferon response element using any method known inthe art.

As an example, but not as a limitation, a plaque assay or quantitativePCR could be used. The growth and replication of the virus with impairedinterferon activity can be compared in the presence and absence of theviral protein to be tested.

In another embodiment the virus administered to the test animal can beany virus with an impaired interferon activity and the protein ofinterest can be any viral protein, the growth and replication of thevirus in the presence of the viral protein is compared to that in theabsence of the viral protein.

The test animal can be monitored for viral titer, or gene expression ofgenes endogenously regulated by interferon or an exogenous gene underthe control of an interferon response element using any method known inthe art. As an example, but not as a limitation, a plaque assay orquantitative PCR could be used. Alternatively, the animal can bemonitored for susceptibility to other infections.

In another preferred embodiment the virus is a chimeric virus with animpaired interferon antagonist activity and an exogenous protein withpotential interferon antagonist activity. As a control additionalanimals could receive a non-chimeric attenuated virus with impairedinterferon antagonist activity.

The test animal can be monitored for viral titer. An increase in viraltiter over that obtained with a control protein or peptide, or noprotein or peptide, would signify that the test protein possessedinterferon antagonist activity. Alternatively, gene expression of genesendogenously regulated by interferon or an exogenous gene under thecontrol of an interferon response element. An increase in geneexpression would signify that the test protein possessed interferonantagonist activity. The animal can also be monitored for susceptibilityto other infections. An increase in susceptibility to other infectionswould indicate that the test protein possessed interferon antagonistactivity.

The screening methods of the invention also encompass identifying thoseviral proteins which antagonize IFN responses. In accordance with thescreeening methods of the invention, induction of IFN responses may bemeasured by assaying levels of IFN expression or expression of targetgenes or reporter genes induced by IFN following transfection with theviral protein or activation of transactivators involved in the IFNexpression and/or the IFN response.

In yet another embodiment of the selection systems of the invention,induction of IFN responses may be determined by measuring thephosphorylated state of components of the IFN pathway followingtransfection with the test viral protein, e.g., IRF-3, which isphosphorylated in response to double-stranded RNA. In response to type IIFN, Jak1 kinase and TyK2 kinase, subunits of the IFN receptor, STAT1,and STAT2 are rapidly tyrosine phosphorylated. Thus, in order todetermine whether the viral protein induces IFN responses, cells, suchas 293 cells, are transfected with the test viral protein and followingtransfection, the cells are lysed. IFN pathway components, such as Jak1kinase or TyK2 kinase, are immunoprecipitated from the infected celllysates, using specific polyclonal sera or antibodies, and the tyrosinephosphorylated state of the kinase is determined by immunoblot assayswith an anti-phosphotyrosine antibody (e.g., see Krishnan et al. 1997,Eur. J. Biochem. 247: 298-305). An enhanced phosphorylated state of anyof the components of the IFN pathway following transfection with theviral protein would indicate induction of IFN responses by the viralprotein.

In yet another embodiment, the screening systems of the inventionencompass measuring the ability to bind specific DNA sequences or thetranslocation of transcription factors induced in response totransfection of a viral protein, e.g., IRF3, STAT1, STAT2, etc. Inparticular, STAT1 and STAT2 are phosphorylated and translocated from thecytoplasm to the nucleus in response to type I IFN. The ability to bindspecific DNA sequences or the translocation of transcription factors canbe measured by techniques known to those of skill in the art, e.g.,electromobility gel shift assays, cell staining, etc.

In yet another embodiment of the screening systems of the invention,induction of IFN responses may be determined by measuring IFN-dependenttranscriptional activation following transfection with the test viralprotein. In this embodiment, the expression of genes known to be inducedby IFN, e.g., Mx, PKR, 2-5-oligoadenylatesynthetase, majorhistocompatibility complex (MHC) class I, etc., can be analyzed bytechniques known to those of skill in the art (e.g., northern blots,western blots, PCR, etc.). Alternatively, test cells such as humanembryonic kidney cells or human osteogenic sarcoma cells, are engineeredto transiently or constitutively express reporter genes such asluciferase reporter gene or chloramphenicol transferase (CAT) reportergene under the control of an interferon stimulated response element,such as the IFN-stimulated promoter of the ISG-54K gene (Bluyssen etal., 1994, Eur. J. Biochem. 220:395-402). Cells are transfected with thetest viral protein and the level of expression of the reporter genecompared to that in untransfected cells or cells transfected with aplasmid lacking a test protein, or alternatively containing a proteinknown not to have interferon antagonist activity. An increase in thelevel of expression of the reporter gene following transfection with thetest viral protein would indicate that the test viral protein isinducing an IFN response.

5.2. SCREENING ASSAYS FOR IDENTIFYING ANTIVIRAL AGENTS THAT TARGET VIRALINTERFERON ANTAGONISTS

The present invention includes methods for screening agents to determineif the agent inhibits or reduces interferon antagonist activity.

The assay utilizes viruses with an impaired interferon antagonistactivity, a plasmid encoding a viral interferon antagonist and a testagent. The assay determines if the test agent inhibits or reducesinterferon antagonist activity (FIG. 3).

Any compound can be screened in connection with the anti-viral assays ofthe present invention, such compounds include, but are not limited to,proteins, polypeptides, peptides, nucleic acids, including dominantnegative mutants, antisense, ribozyme or triple helix molecules,antibodies, small organic molecules, inorganic molecules. In addition,any known antiviral compound can be screened for the ability to inhibitinterferon antagonist activity.

5.2.1. In vitro Screening Assays for Identifying Antiviral Agents thatTarget Viral Interferon Antagonists

The present invention encompasses screening assays that identifyantiviral agents that target a viral interferon antagonist. The presentinvention encompasses screening assays to identify antiviral agents thatmodulate the ability of an interferon antagonist to complement thegrowth and replication of a virus with impaired interferon antagonistactivity. The assay can be performed in any suitable cell that issusceptible to the virus with impaired interferon antagonist activity.

In accordance with the present invention, the virus with impairedinterferon antagonist activity and the interferon antagonist need not beobtained from the same virus. In a preferred embodiment, the virus fromwhich the virus with impaired interferon antagonist activity is derived,is selected based on its ability to infect many types of hosts.

An example of such a virus is influenza virus and an example of such avirus with impaired interferon antagonist activity is delNS1.

Any cell which is susceptible to the virus from which the virus withimpaired interferon activity is derived can be used. Cells may beselected from primary cell cultures, immortalized cells and cell lines.

In accordance with the screening assays of the invention, a test agentmay be assayed for its ability to inhibit or modulate the ability of aninterferon antagonist to complement the replication and growth of avirus with impaired interferon antagonist activity when provided intrans.

In such an embodiment the interferon antagonist may be introduced intothe cell or cell extract.

In another embodiment, the nucleic acids encoding the interferonantagonist may be introduced into the cell. In such an embodiment, thecell may be engineered using standard techniques available to those ofskill in the art to express the interferon antagonist transiently, underinducible conditions or constitutively.

In accordance with the screening assay of the invention, the virus withimpaired interferon antagonist activity may be introduced to the cell orextract as a packaged virion. In yet another embodiment the nucleicacids encoding the virus with impaired interferon antagonist activitymay be introduced into the cell. In such an embodiment, the cell may beengineered using standard techniques available to those of skill in theart to express the nucleic acids encoding the impaired virustransiently, under inducible conditions or constitutively.

In accordance with the present invention, the interferon antagonist andthe impaired virus may be provided consecutively or concurrently in thepresence and absence of a test agent. The screening assays of thepresent invention are not be limited by the order in which thecomponents of the assay are provided to the cell.

In yet another embodiment of the invention, a test agent may be assayedfor its ability to inhibit or modulate the ability of an interferonantagonist to complement the replication and growth of a virus withimpaired interferon antagonist activity when provided in cis. In such anembodiment, a chimeric virus is engineered, such that the interferonantagonist is engineered so that it provides interferon antagonistfunction to a virus that is impaired in this function. The chimericvirus is provided to a cell susceptible to infection by the virus fromwhich the impaired virus is derived. The chimeric virus is provided tothe cell in the presence or absence of the test agent.

Titers are monitored and compared between the treated cells and theuntreated cells, by any method known in the art. Viral titers may bemeasured using any technique known to those of skill in the art.

For example, but not as a limitation titers can be determined by plaqueassay. A lower viral titer in the presence of the test agent as comparedto the absence, would indicate that the test agent possessedanti-interferon antagonist activity and would be a suitable anti-viraldrug candidate.

In a preferred embodiment the virus is influenza virus delNS1. Theinterferon antagonist can be any known interferon antagonist, forexample, but not as a limitation, NS1 of influenza virus and the testagent can be any compound believed to have anti interferon antagonistactivity.

In another embodiment the virus is any virus known to be lackinginterferon antagonist activity. The interferon antagonist can be anyviral interferon antagonist, known or identified by the screening assaysof the present invention, for example, but not as a limitation, NS1 ofinfluenza virus and the test agent can be any compound believed to haveanti interferon antagonist activity.

5.2.2. In vivo Screening Assays for Identifying Antiviral Agents thatTarget Viral Interferon Antagonists

The present invention encompasses screening assays that identifyantiviral agents that target a viral interferon antagonist. The presentinvention encompasses screening assays to identify antiviral agents thatmodulate the ability of an interferon antagonist to complement thegrowth and replication of a virus with impaired interferon antagonistactivity. The assay can be performed in any suitable animal that issusceptible to the virus with impaired interferon antagonist activity.

In accordance with the present invention, the virus with impairedinterferon antagonist activity and the interferon antagonist need not beobtained from the same virus. In a preferred embodiment, the virus fromwhich the virus with impaired interferon antagonist activity is derived,is selected based on its ability to infect many types of hosts. Anexample of such a virus is influenza virus and an example of such avirus with impaired interferon antagonist activity is delNS1.

Any animal which is susceptible to the virus from which the virus withimpaired interferon activity is derived can be used. As an example, butnot as a limitation, avians, monkeys, rats mice, dogs, rabbits, or pigsmay be used.

In accordance with the screening assays of the invention, a test agentmay be assayed for its ability to inhibit or modulate the ability of aninterferon antagonist to complement the replication and growth of avirus with impaired interferon antagonist activity when provided intrans. In such an embodiment the interferon antagonist may be introducedinto the animal. In another embodiment, the nucleic acids encoding theinterferon antagonist may be introduced into the animal. In such anembodiment, the animal may be engineered using standard techniquesavailable to those of skill in the art to express the interferonantagonist transiently, under inducible conditions or constitutively.

In accordance with the screening assay of the invention, the virus withimpaired interferon antagonist activity may be introduced to the animalas a packaged virion. In yet another embodiment the nucleic acidsencoding the virus with impaired interferon antagonist activity may beintroduced into the animal. In such an embodiment, the cell may beengineered using standard techniques available to those of skill in theart to express the nucleic acids encoding the impaired virustransiently, under inducible conditions or constitutively.

In accordance with the present invention, the interferon antagonist andthe impaired virus may be provided consecutively or concurrently in thepresence and absence of a test agent. The screening assays of thepresent invention are not be limited by the order in which thecomponents of the assay are provided to the animal.

In yet another embodiment of the invention, a test agent may be assayedfor its ability to inhibit or modulate the ability of an interferonantagonist to complement the replication and growth of a virus withimpaired interferon antagonist activity when provided in cis. In such anembodiment, a chimeric virus is engineered, such that the interferonantagonist is engineered so that it provides interferon antagonistfunction to a virus that is impaired in this function.

The chimeric virus is provided to an animal susceptible to infection bythe virus from which the impaired virus is derived. The chimeric virusis provided to the animal in the presence or absence of the test agent.

Titers are monitored and compared between the treated animals and theuntreated animals, by any method known in the art. Viral titers may bemeasured using any technique known to those of skill in the art. Forexample, but not as a limitation titers can be determined by plaqueassay. A lower viral titer in the presence of the test agent as comparedto its absence, would indicate that the test agent possessedanti-interferon antagonist activity and would be a suitable anti-viraldrug candidate.

In another embodiment the virus is any virus known to be lackinginterferon antagonist activity. The interferon antagonist can be anyviral interferon antagonist, known or identified by the screening assaysof the present invention, for example, but not as a limitation, NS1 ofinfluenza virus and the test agent can be any compound believed to haveanti interferon antagonist activity.

5.3. VIRUSES WITH IMPAIRED INTERFERON ANTAGONIST ACTIVITY

The screening assays of the invention can be used to identify viralproteins with interferon antagonist activity. Once such a viral proteinhas been identified, the protein, the nucleic acid encoding the proteinand the elements regulating the expression of the protein can be thetarget of manipulation and/or mutation to create a virus with impairedinterferon antagonist activity.

Viruses with impaired interferon antagonist activity can includenaturally occurring mutants with impaired interferon antagonistactivity, engineered mutants with impaired interferon antagonistactivity and recombinant viruses with impaired interferon antagonistactivity.

Any mutant virus or strain which has a decreased IFN antagonist activitycan be selected and used in accordance with the invention. In oneembodiment, naturally occurring mutants or variants, or spontaneousmutants can be selected that have an impaired ability to antagonize thecellular IFN response. In another embodiment, mutant viruses can begenerated by exposing the virus to mutagens, such as ultravioletirradiation or chemical mutagens, or by multiple passages and/or passagein non-permissive hosts. Screening in a differential growth system canbe used to select for those mutants having impaired IFN antagonistfunction. For viruses with segmented genomes, the attenuated phenotypecan be transferred to another strain having a desired antigen byreassortment, (i.e., by coinfection of the attenuated virus and thedesired strain, and selection for reassortants displaying bothphenotypes).

In another embodiment, mutations can be engineered into a negativestrand RNA virus such as influenza, RSV, NDV, VSV and PIV, using“reverse genetics” approaches. In this way, natural or other mutationswhich confer the attenuated phenotype can be engineered into vaccinestrains. For example, deletions, insertions or substitutions of thecoding region of the gene responsible for IFN antagonist activity (suchas the NS1 of influenza) can be engineered. Deletions, substitutions orinsertions in the non-coding region of the gene responsible for IFNantagonist activity are also contemplated. To this end, mutations in thesignals responsible for the transcription, replication, polyadenylationand/or packaging of the gene responsible or the IFN-antagonist activitycan be engineered. For example, in influenza, such modifications caninclude but are not limited to: substitution of the non-coding regionsof an influenza A virus gene by the non-coding regions of an influenza Bvirus gene (Muster, et al., 1991, Proc. Natl. Acad. Sci. USA, 88:5177),base pairs exchanges in the non-coding regions of an influenza virusgene (Fodor, et al., 1998, J Virol. 72:6283), mutations in the promoterregion of an influenza virus gene (Piccone, et al., 1993, Virus Res.28:99; Li, et al., 1992, J Virol. 66:4331), substitutions and deletionsin the stretch of uridine residues at the 5′ end of an influenza virusgene affecting polyadenylation (Luo, et al., 1991, J Virol. 65:2861; Li,et al., J Virol. 1994, 68(2):1245-9). Such mutations, for example to thepromoter, could down-regulate the expression of the gene responsible forIFN antagonist activity. Mutations in viral genes which may regulate theexpression of the gene responsible for IFN antagonist activity are alsowithin the scope of viruses that can be used in accordance with theinvention.

The present invention also relates to mutations to the NS1 gene segmentthat may not result in an altered IFN antagonist activity or anIFN-inducing phenotype but rather results in altered viral functions andan attenuated phenotype e.g., altered inhibition of nuclear export ofpoly(A)-containing mRNA, altered inhibition of pre-mRNA splicing,altered inhibition of the activation of PKR by sequestering of dsRNA,altered effect on translation of viral RNA and altered inhibition ofpolyadenylation of host mRNA (e.g., see Krug in Textbook of Influenza,Nicholson et al. Ed. 1998, 82-92, and references cited therein).

The reverse genetics technique involves the preparation of syntheticrecombinant viral RNAs that contain the non-coding regions of thenegative strand virus RNA which are essential for the recognition byviral polymerases and for packaging signals necessary to generate amature virion. The recombinant RNAs are synthesized from a recombinantDNA template and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells. A more efficient transfection is achieved if theviral polymerase proteins are present during transcription of thesynthetic RNAs either in vitro or in vivo. The synthetic recombinantRNPs can be rescued into infectious virus particles. The foregoingtechniques are described in U.S. Pat. No. 5,166,057 issued Nov. 24,1992; in U.S. Pat. No. 5,854,037 issued Dec. 29, 1998; in EuropeanPatent Publication EP 0702085A1, published Feb. 20, 1996; in U.S. Pat.No. 6,146,642; in International Patent Publications PCT WO97/12032published Apr. 3, 1997; WO96/34625 published Nov. 7, 1996; in EuropeanPatent Publication EP-A780475; WO 99/02657 published Jan. 21, 1999; WO98/53078 published Nov. 26, 1998; WO 98/02530 published Jan. 22, 1998;WO 99/15672 published Apr. 1, 1999; WO 98/13501 published Apr. 2, 1998;WO 97/06270 published Feb. 20, 1997; and EPO 780 47SA1 published Jun.25, 1997, each of which is incorporated by reference herein in itsentirety.

Attenuated viruses generated by the reverse genetics approach can beused in the vaccine and pharmaceutical formulations described herein.Reverse genetics techniques can also be used to engineer additionalmutations to other viral genes important for vaccine production—i.e.,the epitopes of useful vaccine strain variants can be engineered intothe attenuated virus. Alternatively, completely foreign epitopes,including antigens derived from other viral or non-viral pathogens canbe engineered into the attenuated strain. For example, antigens ofnon-related viruses such as HIV (gp160, gp120, gp41) parasite antigens(e.g., malaria), bacterial or fungal antigens or tumor antigens can beengineered into the attenuated strain. Alternatively, epitopes whichalter the tropism of the virus in vivo can be engineered into thechimeric attenuated viruses of the invention.

In an alternate embodiment, a combination of reverse genetics techniquesand reassortant techniques can be used to engineer attenuated viruseshaving the desired epitopes in segmented RNA viruses. For example, anattenuated virus (generated by natural selection, mutagenesis or byreverse genetics techniques) and a strain carrying the desired vaccineepitope (generated by natural selection, mutagenesis or by reversegenetics techniques) can be co-infected in hosts that permitreassortment of the segmented genomes. Reassortants that display boththe attenuated phenotype and the desired epitope can then be selected.

In another embodiment, the virus to be mutated is a DNA virus (e.g.,vaccinia, adenovirus, baculovirus) or a positive strand RNA virus (e.g.,polio virus). In such cases, recombinant DNA techniques which are wellknown in the art may be used (e.g., see U.S. Pat. No. 4,769,330 toPaoletti, U.S. Pat. No. 4,215,051 to Smith each of which is incorporatedherein by reference in its entirety).

Any virus may be engineered in accordance with the present invention,including but not limited to the families set forth in Table 1 below.TABLE 1 FAMILIES OF HUMAN AND ANIMAL VIRUSES VIRUS CHARACTERISTICS VIRUSFAMILY dsDNA  Enveloped Poxviridae Irididoviridae Herpesviridae Nonenveloped Adenoviridae Papovaviridae Hepadnaviridae ssDNA Nonenveloped Parvoviridae dsRNA  Nonenveloped Reoviridae BirnaviridaessRNA  Enveloped   Positive-Sense Genome    No DNA Step in ReplicationTogaviridae Flaviviridae Coronaviridae Hepatitis C Virus    DNA Step inReplication Retroviridae   Negative-Sense Genome    Non-Segmented GenomeParamyxoviridae Rhabdoviridae Filoviridae    Segmented GenomeOrthomyxoviridae Bunyaviridae Arenaviridae Nonenveloped PicornaviridaeCaliciviridaeAbbreviations used:ds = double stranded;ss = single stranded;enveloped = possessing an outer lipid bilayer derived from the host cellmembrane;positive-sense genome = for RNA viruses, genomes that are composed ofnucleotide sequences that are directly translated onribosomes, = for DNA viruses, genomes that are composed of nucleotidesequences that are the same as the mRNA;negative-sense genome = genomes that are composed of nucleotidesequences complementary to the positive-sense strand.

Antisense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules, as discussed above. These includetechniques for chemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

5.4. VACCINE FORMULATIONS

The present invention encompasses screening methods to identify viralproteins with interferon antagonist activities, such as influenza virusNS1, ebola virus VP35 and respiratory syncytial virus NS2. Once suchinterferon antagonist viral proteins have been identified they can betargeted in the virus for mutation or manipulation to create a viruswith an impaired interferon antagonist and an attenuated phenotype.While the present invention provides examples of interferon antagonistactivities for influenza virus, ebola virus, and respiratory syncytialvirus, these are provided by way of example and not limitation. However,the principles of the invention can be analogously applied andextrapolated to other viruses, including other segmented andnon-segmented RNA viruses, such viruses may include, but are not limitedto paramyxoviruses (Sendai virus, parainfluenza virus, mumps, Newcastledisease virus) morbillivirus (measles virus, canine distemper virus, andrinderpest virus); pneumovirus (respiratory syncytial virus and bovinerespiratory virus); rhabdovirus (vesicular stomatis virus andlyssavirus); lentiviruses, including human immunodeficiency virus (HIV),RNA viruses including hepatitis C virus and retroviruses includinghepatitis B virus and HIV, and DNA viruses, including adenovirus, adenoassociated virus, hepadna viruses, herpes viruses and poxvirus.

The invention encompasses vaccine formulations comprising attenuatedviruses having an impaired ability to antagonize the cellular IFNresponse, and a suitable excipient. The virus used in the vaccineformulation may be selected from naturally occurring mutants orvariants, mutagenized viruses or genetically engineered viruses.Attenuated strains of segmented RNA viruses can also be generated viareassortment techniques, or by using a combination of the reversegenetics approach and reassortment techniques. Naturally occurringvariants include viruses isolated from nature as well as spontaneousoccurring variants generated during virus propagation, having animpaired ability to antagonize the cellular IFN response. The attenuatedvirus can itself be used as the active ingredient in the vaccineformulation. Alternatively, the attenuated virus can be used as thevector or “backbone” of recombinantly produced vaccines. To this end,recombinant techniques such as reverse genetics (or, for segmentedviruses, combinations of the reverse genetics and reassortmenttechniques) may be used to engineer mutations or introduce foreignantigens into the attenuated virus used in the vaccine formulation. Inthis way, vaccines can be designed for immunization against strainvariants, or in the alternative, against completely different infectiousagents or disease antigens.

Virtually any heterologous gene sequence may be constructed into theviruses of the invention for use in vaccines. Preferably, epitopes thatinduce a protective immune response to any of a variety of pathogens, orantigens that bind neutralizing antibodies may be expressed by or aspart of the viruses. For example, heterologous gene sequences that canbe constructed into the viruses of the invention for use in vaccinesinclude but are not limited to epitopes of human immunodeficiency virus(HIV) such as gp120; hepatitis B virus surface antigen (HBsAg); theglycoproteins of herpes virus (e.g. gD, gE); VP1 of poliovirus;antigenic determinants of non-viral pathogens such as bacteria andparasites, to name but a few. In another embodiment, all or portions ofimmunoglobulin genes may be expressed. For example, variable regions ofanti-idiotypic immunoglobulins that mimic such epitopes may beconstructed into the viruses of the invention. In yet anotherembodiment, tumor associated antigens may be expressed.

Either a live recombinant viral vaccine or an inactivated recombinantviral vaccine can be formulated. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

Vaccine formulations may include genetically engineered negative strandRNA viruses that have mutations in the NS1 or analogous gene includingbut not limited to the truncated NS1 influenza mutants described in theworking examples, infra. They may also be formulated using naturalvariants, such as the A/turkey/Ore/71 natural variant of influenza A, orB/201, and B/AWBY-234, which are natural variants of influenza B. Whenformulated as a live virus vaccine, a range of about 104 pfu to about5×10⁶ pfu per dose should be used.

Many methods may be used to introduce the vaccine formulations describedabove, these include but are not limited to intranasal, intratracheal,oral, intradermal, intramuscular, intraperitoneal, intravenous, andsubcutaneous routes. It may be preferable to introduce the virus vaccineformulation via the natural route of infection of the pathogen for whichthe vaccine is designed, or via the natural route of infection of theparental attenuated virus. Where a live influenza virus vaccinepreparation is used, it may be preferable to introduce the formulationvia the natural route of infection for influenza virus. The ability ofinfluenza virus to induce a vigorous secretory and cellular immuneresponse can be used advantageously. For example, infection of therespiratory tract by influenza viruses may induce a strong secretoryimmune response, for example in the urogenital system, with concomitantprotection against a particular disease causing agent.

A vaccine of the present invention, comprising 10⁴-5×10⁶ pfu of mutantviruses with altered IFN antagonist activity, could be administeredonce. Alternatively, a vaccine of the present invention, comprising10⁴-5×10⁶ pfu of mutant viruses with altered IFN antagonist activity,could be administered twice or three times with an interval of 2 to 6months between doses. Alternatively, a vaccine of the present invention,comprising 10⁴-5×10⁶ pfu of mutant viruses with altered IFN antagonistactivity, could be administered as often as needed to an animal,preferably a mammal, and more preferably a human being.

The invention encompasses vaccine formulations comprised of anattenuated virus wherein the attenuation results from a mutation in agene encoding an interferon antagonist.

The invention also encompasses vaccine formulations comprised of anattenuated virus wherein the attenuation results from a mutation in agene encoding an interferon antagonist in combination with one or moremutations in other viral genes.

The invention also includes vaccine formulations which are chimericviruses. A chimeric virus could be comprised of any virus where theinterferon antagonist gene is derived from either a different virus or adifferent strain of the same virus. By way of example, but not alimitation a chimeric virus could include an influenza A virus whereinthe NS1 gene has been replaced by VP35 from ebola virus. The VP35 genecould contain a mutation which results in an attenuated phenotype of thechimeric virus.

In a preferred embodiment the attenuated virus is respiratory syncytialvirus with a mutation in the NS2 gene. An attenuated ebola virus with amutation in the VP35 would comprise another preferred embodiment. Inanother preferred embodiment, the attenuated virus is influenza A viruswith a mutation in the NS1 gene.

The invention includes a vaccine formulation comprising an attenuatedvirus for treating or preventing any infectious disease. The infectiousdisease could be a virus. By way of example, but not as a limitation thevaccine formulation could be used to treat or prevent infection withinfluenza virus, ebola virus, respiratory syncytial virus, HIV, herpesvirus, hepatitis C virus or hepatitis B virus. The infectious diseasecould consist of a bacterium or a parasite. Additionally the vaccinecould be used to treat or prevent cancer or tumor growth.

5.5. PHARMACEUTICAL COMPOSITIONS

The present invention encompasses pharmaceutical compositions comprisinganti-viral agents which are identified by the screening assays describedherein to inhibit or modulate viral interferon antagonist activities.

The mutant IFN-inducing viruses of the invention may be engineered usingthe methods described herein to express proteins or peptides which wouldtarget the viruses to a particular site. In a preferred embodiment, theIFN-inducing viruses would be targeted to the site of the infection orthe site of entry of the infectious agent. In such an embodiment, themutant viruses can be engineered to express the antigen combining siteof an antibody which recognized the cellular receptor for the infectiouspathogen, thus targeting the IFN-inducing virus to the site of theinfection. Thus, in accordance with the invention, the IFN-inducingviruses may be engineered to express any target gene product, includingpeptides, proteins, such as enzymes, hormones, growth factors, antigensor antibodies, which will function to target the virus to a site in needof anti-viral, antibacterial, anti-microbial or anti-cancer activity.

The invention provides methods for the treatment or prevention of viralinfections in an animal, preferably a mammal and most preferably ahuman, said methods comprising the administration of a therapeuticallyor prophylactically effective amount of an anti-interferon antagonist ornucleic acid molecules encoding said anti-interferon antagonist.Examples of viral infections which can be treated or prevented inaccordance with this invention include, but are limited to, viralinfections caused by retroviruses (e.g., human T-cell lymphotrophicvirus (HTLV) types I and II and human immunodeficiency virus (HIV)),herpes viruses (e.g., herpes simplex virus (HSV) types I and II,Epstein-Barr virus and cytomegalovirus), arenaviruses (e.g., lassa fevervirus), paramyxoviruses (e.g., morbillivirus virus, human respiratorysyncytial virus, and pneumovirus), adenoviruses, bunyaviruses (e.g.,hantavirus), cornaviruses, filoviruses (e.g., Ebola virus), flaviviruses(e.g., hepatitis C virus (HCV), yellow fever virus, and Japaneseencephalitis virus), hepadnaviruses (e.g., hepatitis B viruses (HBV)),orthomyoviruses (e.g., Sendai virus and influenza viruses A, B and C),papovaviruses (e.g., papillomavirues), picornaviruses (e.g.,rhinoviruses, enteroviruses and hepatitis A viruses), poxviruses,reoviruses (e.g., rotavirues), togaviruses (e.g., rubella virus), andrhabdoviruses (e.g., rabies virus). The treatment and/or prevention of aviral infection includes, but is not limited to, alleviating one or moresymptoms associated with said infection, the inhibition, reduction orsuppression of viral replication, and/or the enhancement of the immuneresponse.

Compounds identified through assays described, above, in Section 5.1 and5.2, which inhibit interferon antagonists by decreasing the expressionand/or activity of interferon antagonists can be used in accordance withthe invention to prevent or treat symptoms associated with viralinfections. Further, inhibitors of interferon antagonists can be used totreat viral infections. As discussed above, such compounds can include,but are not limited to nucleic acids, proteins, peptides,phosphopeptides, small organic or inorganic molecules, or antibodies(including, for example, polyclonal, monoclonal, human, humanized,anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)₂and Fab expression library fragments, and epitope-binding fragmentsthereof).

In a specific embodiment, interferon antagonists or fragmentsrepresenting a functional domain of interferon antagonists areadministered to an animal at sufficient dosages such that interferonantagonists activity is decreased in vivo, e.g., by mimicking thefunction of interferon antagonists in vivo.

The proteins and peptides which may be used in such methods includesynthetic (e.g., recombinant or chemically synthesized) proteins andpeptides, as well as naturally occurring proteins and peptides. Theproteins and peptides may have both naturally occurring and/ornon-naturally occurring amino acid residues (e.g., D-amino acidresidues) and/or one or more non-peptide bonds (e.g., imino, ester,hydrazide, semicarbazide, and azo bonds). The proteins or peptides mayalso contain additional chemical groups (e.g., functional groups)present at the amino and/or carboxy termini, such that, for example, thestability, bioavailability, and/or inhibitory activity of the peptide isenhanced. Exemplary functional groups include hydrophobic groups (e.g.,carbobenzoxyl, dansyl, and t-butyloxycarbonyl groups) an acetyl group, a9-fluorenylmethoxy-carbonyl group, and macromolecular carrier groups(e.g., lipid-fatty acid conjugates, polyethylene glycol, orcarbohydrates) including peptide groups.

In instances wherein the compound to be administered is a peptidecompound, DNA sequences encoding the peptide compound can be directlyadministered to an animal. Any of the techniques discussed, below, whichachieve intracellular administration of compounds, such as, for example,liposome administration, can be utilized for the administration of suchDNA molecules. The DNA molecules can be produced, for example, by wellknown recombinant techniques.

In certain embodiments, a composition of the invention is administeredto an animal to ameliorate one or more symptoms associated with a viralinfection or a disease or disorder resulting, directly or indirectly,from a viral infection. In a specific embodiment, a composition of theinvention is administered to a human to ameliorate one or more symptomsassociated with AIDS. In certain other embodiments, a composition of theinvention is administered to reduce the titer of a virus in an animal.In certain other embodiments, a composition of the invention isadministered to an animal to enhance or promote the immune response.

In a specific embodiment, a composition comprising a therapeuticallyeffective amount of one or more anti-interferon antagonist isadministered to an animal in order to ameliorate one or more symptomsassociated with a viral infection. In another embodiment, a compositioncomprising a therapeutically effective amount of one or moreanti-interferon antagonist is administered to an animal in order toreduce the titer of a virus in an animal. In another embodiment, acomposition comprising a therapeutically effective amount of one or moreanti-interferon antagonist and one or more antibodies immunospecific forone or more viral antigens is administered to an animal in order toameliorate one or more symptoms associated with a viral infection. Inyet another embodiment, a composition comprising a therapeuticallyeffective amount of one or more anti-interferon antagonist and one ormore antibodies immunospecific for one or more viral antigens isadministered to an animal in order to reduce the titer of a virus in ananimal.

Anti-interferon antagonist may be administered alone or in combinationwith other types of anti-viral agents. Examples of anti-viral agentsinclude, but are not limited to: cytokines (e.g., IFN-α, IFN-β, andIFN-δ); inhibitors of reverse transcriptase (e.g., AZT, 3TC, D4T, ddC,ddI, d4T, 3TC, adefovir, efavirenz, delavirdine, nevirapine, abacavir,and other dideoxynucleosides or dideoxyfluoronucleosides); inhibitors ofviral mRNA capping, such as ribavirin; inhibitors of proteases such HIVprotease inhibitors (e.g., amprenavir, indinavir, nelfinavir, ritonavir,and saquinavir,); amphotericin B; castanospermine as an inhibitor ofglycoprotein processing; inhibitors of neuraminidase such as influenzavirus neuraminidase inhibitors (e.g., zanamivir and oseltamivir);topoisomerase I inhibitors (e.g., camptothecins and analogs thereof);amantadine; and rimantadine. Such anti-viral agents may be administeredto an animal, preferably a mammal and most preferably a human, for theprevention or treatment of a viral infection prior to (e.g., 1 minute,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to(e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after),or concomitantly with the administration of anti-interferon antagonistto the animal.

In a specific embodiment, one or more anti-interferon antagonist areadministered to an animal, preferably a mammal and most preferably ahuman, for the prevention or treatment of a viral infection prior to(e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before),subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or1 week after), or concomitantly with the administration of plasma to theanimal.

In a preferred embodiment, one or more anti-interferon antagonist areadministered to an animal, preferably a mammal and most preferably ahuman, for the prevention or treatment of a viral infection prior to(e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before),subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or1 week after), or concomitantly with the administration of IgGantibodies, IgM antibodies and/or one or more complement components tothe animal. In another preferred embodiment, anti-interferon antagonistare administered to an animal, preferably a mammal and most preferably ahuman, for the prevention or treatment of a viral infection prior to(e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before),subsequent to (e.g., 1 minute, 15 minutes, 30 minutes,.45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or1 week after), or concomitantly with the administration of antibodiesimmunospecific for one or more viral antigens. Example of antibodiesimmunospecific for viral antigens include, but are not limited to,Synagis®, PRO542, Ostavir, and Protovir.

Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The pharmaceutical compositions of thepresent invention may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local. In addition, ina preferred embodiment it may be desirable to introduce thepharmaceutical compositions of the invention into the lungs by anysuitable route. Pulmonary administration can also be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger & Peppas, 1983, J. Macromol. Sci.Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190;During et al., 1989, Ann. Neurol. 25:351 (1989); Howard et al., 1989, J.Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, i.e., thelung, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, 1984, in Medical Applications of Controlled Release, supra,vol. 2, pp. 115-138). Other controlled release systems are discussed inthe review by Langer (1990, Science 249:1527-1533).

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of an attenuated virus, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeiae for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the pharmaceuticalcomposition is administered. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. These compositions can beformulated as a suppository. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the Therapeutic,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

The amount of the pharmaceutical composition of the invention which willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges foradministration are generally about 10⁴-5×10⁶ pfu and can be administeredonce, or multiple times with intervals as often as needed.Pharmaceutical compositions of the present invention comprising10⁴-5×10⁶ pfu of mutant viruses with altered IFN antagonist activity,can be administered intranasally, intratracheally, intramuscularly orsubcutaneously. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The invention includes a pharmaceutical composition comprising anattenuated virus with an impairment in the interferon antagonistactivity. The invention also includes a pharmaceutical compositioncomprising an attenuated virus with an impairment in the interferonantagonist activity wherein the attenuated virus is a chimeric virus. Achimeric virus could be comprised of any virus where the interferonantagonist gene is derived from either a different virus or a differentstrain of the same virus. By way of example, but not a limitation achimeric virus could include an influenza A virus wherein the NS1 genehas been replaced by VP35 from ebola virus. The VP35 gene could containa mutation which results in an attenuated phenotype of the chimericvirus.

The invention also includes pharmaceutical compositions comprising ananti-viral agent identified by the assays described herein. Saidanti-virals would target the viral gene protein that antagonizesinterferon function. The anti-viral could be comprised of a protein orpeptide, an amino acid, an anti-sense molecule, a ribozyme, any smallorganic or inorganic molecule.

Methods of introduction of the ant-viral agent include but are notlimited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The pharmaceuticalcompositions of the present invention may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, in a preferred embodiment it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the lungs by any suitable route. Pulmonary administration can alsobe employed, e.g., by use of an inhaler or nebulizer, and formulationwith an aerosolizing agent.

Any viral infection could be treated with the ant-viral agent, providedthat the viral etiological agent contains an interferon antagonist thatis sensitive to the anti-viral agent. By way of example, but not by wayof limitation, the viral infections that could be treated with ananti-viral agent that targets the interferon antagonist would includeinfluenza virus, respiratory syncytial virus, and ebola virus.

5.6. DEMONSTRATION OF THERAPEUTIC/PROPHYLACTIC UTILITY OF COMPOSITIONSOF THE INVENTION

The present invention encompasses pharmaceutical compositions comprisinganti-viral agents which are identified by the screening assays describedherein to inhibit or modulate viral interferon antagonist activities.

The present invention also encompasses pharmaceutical compositionscomprising mutant viruses with altered IFN antagonist activity to beused as anti-viral agents. The pharmaceutical compositions, of thepresent invention, have utility as an anti-viral prophylactic and may beadministered to an individual at risk of getting infected or is expectedto be exposed to a virus. For example, in the event that a child comeshome from school where he is exposed to several classmates with the flu,a parent would administer the anti-viral pharmaceutical composition ofthe invention to herself, the child and other family members to preventviral infection and subsequent illness. People traveling to parts of theworld where a certain infectious disease is prevalent (e.g. hepatitis Avirus, malaria, etc.) can also be treated.

The compositions of the invention are preferably tested in vitro, andthen in vivo for the desired therapeutic or prophylactic activity, priorto use in humans. For example, in vitro assays to demonstrate thetherapeutic or prophylactic utility of a composition include, the effectof a composition on a cell line, particularly one characteristic of aspecific type of cancer, or a patient tissue sample. The effect of thecomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Testcompositions can be tested for their ability to augment activated immunecells by contacting activated immune cells with a test composition or acontrol composition and determining the ability of the test compositionto modulate the biological activity of the activated immune cells. Theability of a test composition to modulate the biological activity ofactivated immune cells can be assessed by detecting the expression ofcytokines or antigens, detecting the proliferation of immune cells,detecting the activation of signaling molecules, detecting the effectorfunction of immune cells, or detecting the differentiation of immunecells. Techniques known to those of skill in the art can be used formeasuring these activities. For example, cellular proliferation can beassayed by ³H-thymidine incorporation assays and trypan blue cellcounts. Cytokine and antigen expression can be assayed, for example, byimmunoassays including, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,immunohistochemistry radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoprecipitationassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays and FACS analysis. The activation of signaling moleculescan be assayed, for example, by kinase assays and electromobility shiftassays (EMSAs). The effector function of T-cells can be measured, forexample, by a 51Cr-release assay (see, e.g., Palladino et al., 1987,Cancer Res. 47:5074-5079 and Blachere et al., 1993, J. Immunotherapy14:352-356).

Test composition can be tested for their ability to reduce tumorformation in patients (i.e., animals) suffering from cancer. Testcompositions can also be tested for their ability to reduce viral loador bacterial numbers in vitro and in vivo (e.g., in patients sufferingfrom an infectious disease) utilizing techniques known to one of skillin the art. Test compositions can also be tested for their ability toalleviate of one or more symptoms associated with cancer or aninfectious disease (e.g., a viral or microbial infection). Testcompositions can also be tested for their ability to decrease the timecourse of the infectious disease

Therapeutic and or prophylactic utility, of the present invention can bedemonstrated by way of an in vitro or an in vivo assay. In vitro assayscould be performed in any cell line. The cell line could be derived froman animal, insect or plant. Preferably it is derived from an animal andmost preferably it is derived from a mammal. Examples of such cell linesinclude, but are not limited to MDCK, HeLa, Cos, and NIH3T3 cells. Invivo assays could be performed in any animal infected with the pathogenof interest. Preferably the animal would be a mammal.

In vitro assays would include any assay that measures the infectiousburden of a given pathogen. For example viral load could be measured byany assay known in the art. By way of example, but not as a limitation,a plaque assay or HA assay, or quantitative PCR assay or branched DNAassay could be used.

Infectious burden could be monitored in an in vivo assay by any methodknown in the art including those described above as well as by methodsof histology and microscopy. These assays are offered merely as examplesand are not intended to be a limitation.

The present invention also provides assays for use in drug discovery inorder to identify or verify the efficacy of compounds for treatment orprevention of an infectious disease. Candidate compounds can be assayedfor their ability to modulate infectious burden in a subject having aninfectious disease. Compounds able to lower the infectious burden in asubject having an infectious disease can be used as lead compounds forfurther drug discovery, or used therapeutically. Infectious burden canbe assayed by immunoassays, gel electrophoresis, plaque assay or anyassay that measures viral burden or any other method taught herein orknown to those skilled in the art. Such assays can be used to screencandidate drugs, in clinical monitoring or in drug development, wherelevel of infectious burden can serve as a surrogate marker for clinicaldisease.

In various specific embodiments, in vitro assays can be carried out withcells representative of cell types involved in a disorder, to determineif a compound has a desired effect upon such cell types. For example,HeLa cells or Vero cells can be used to determine if a compound has adesired effect upon such cells.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to rats,mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, priorto administration to humans, any animal model system known in the artmay be used. It is also apparent to the skilled artisan that, based uponthe present disclosure, transgenic animals can be produced with“knock-out” mutations of the gene or genes encoding any cellularfunction required by the infectious pathogen or alternatively any immunefunction that allows the host animal to mount an effective immuneresponse against an infectious pathogen. A “knock-out” mutation of agene is a mutation that causes the mutated gene to not be expressed, orexpressed in an aberrant form or at a low level, such that the activityassociated with the gene product is nearly or entirely absent.Preferably, the transgenic animal is a mammal, more preferably, thetransgenic animal is a mouse.

In one embodiment, candidate compounds that modulate the level ofinfectious burden are identified or verified in human subjects sufferingfrom said infectious disease. In accordance with this embodiment, acandidate compound or a control compound is administered to the humansubject, and the effect of a test compound on infectious burden isdetermined by analyzing the level of the infectious pathogen or the mRNAencoding the same in a biological sample (e.g., serum or plasma). Acandidate compound that alters the level of the infectious pathogen canbe identified by comparing the level of the infectious pathogen or mRNAencoding the same in a subject or group of subjects treated with acontrol compound to that in a subject or group of subjects treated witha candidate compound. Alternatively, alterations in the infectiousburden can be identified by comparing the level of the infectiouspathogen or mRNA encoding the same in a subject or group of subjectsbefore and after the administration of a candidate compound. Techniquesknown to those of skill in the art can be used to obtain the biologicalsample and analyze the MRNA or protein expression.

In another embodiment, candidate compounds that modulate the level ofinfectious burden are identified or verified in human subjects havingsaid infectious disease. In accordance with this embodiment, a candidatecompound or a control compound is administered to the human subject, andthe effect of a candidate compound on the level of the infectiouspathogen is determined. A candidate compound that alters the level ofinfectious burden of the infectious pathogen can be identified bycomparing biological samples from subjects treated with a controlcompound to samples from subjects treated with the candidate compound.Techniques known to those of skill in the art can be used to detectchanges in the level of infectious burden, changes or changes in acellular response to an infectious pathogen. For example, RT-PCR orimmunoprecipitation followed by western blot analysis can be used todetect changes in the level of infectious burden.

In another embodiment, candidate compounds that reduce the severity ofone or more symptoms associated with an infectious pathogen areidentified in human subjects having said infectious pathogen. Inaccordance with this embodiment, a candidate compound or a controlcompound is administered to a human subject, suffering from aninfectious pathogen and the effect of a candidate compound on one ormore symptoms of the infectious pathogen is determined. A candidatecompound that reduces one or more symptoms can be identified bycomparing the subjects treated with a control compound to the subjectstreated with the test compound. Techniques known to physicians familiarwith infectious diseases can be used to determine whether a candidatecompound reduces one or more symptoms associated with the infectiousdisease.

5.7. DEMONSTRATION OF THE ABILITY OF VIRAL INTERFERON ANTAGONISTS TOENHANCE TRANSLATION

The present invention relates to the ability of viral interferonantagonists to enhance translation of mRNAs. The interferon antagonistsidentified by the screening assays of the present invention have utilityin in vitro and in vivo protocols to enhance levels of translation. Suchin vitro and in vivo protocols may include: (1) enhancing levels oftranslation in assay systems where enhanced translation levels arerequired, such as reporter assay systems where enhanced sensitivity isrequired; (2) enhancing levels of translation in cell based assays toincrease detection of a target protein; (3) ex vivo based gene therapyprotocols, to increase detection of a marker or increase expression of atarget gene; and (4) in vivo based gene therapy protocols to increasedetection of a marker or increase expression of a target gene.

The in vitro and in vivo protocols involving the use of interferonantagonists to enhance levels of translation encompass research basedassays, highthroughput screening assays, drug screening assays, invitro, ex vivo, and in vivo diagnostic, prophylactic and therapeuticsassays and protocols.

Any interferon antagonist identified by the assays of the presentinvention may be used in this embodiment. In a preferred embodiment, theinterferon antagonist used to enhance translation is NS2 of respiratorysyncytial virus or VP35 of ebola virus. In a most preferred embodiment,the interferon antagonist is the NS1 protein of influenza A virus.

5.8. KITS

The present invention provides for kits that can be used in the abovemethods. In one embodiment the kit would be comprised of a virus,contained in an appropriate package, with impaired interferon antagonistactivity. As an example, but not as a limitation the delNS1 influenza Avirus mutant could be used. The kit would also contain a positivecontrol, in an appropriate package, consisting of a viral interferonantagonist. By way of example, but not as a limitation the viralinterferon antagonist could include NS2 of respiratory syncytial virus,VP35 of ebola virus or NS1 of influenza A virus. The kit would alsocontain a negative control. The kit would also contain an appropriateplasmid or vector to express the positive and or negative control. Alsoincluded in the kit would be a reporter construct, in an appropriatepackage, that is linked to an interferon responsive element. Thereporter construct could be the luciferase gene for example, but not asa limitation. The kit would also contain instructions for use.

6. EXAMPLE Transfection of Viral Interferon Antagonists ComplementsGrowth of Influenza delNS1 Virus: A Method to Identify Novel InterferonAntagonists

The following example demonstrates the use of a virus with impairedinterferon antagonist activity, such as influenza delNS1 virus, toscreen for viral proteins with interferon antagonist activities. Theexample describes the use of such an impaired virus to assay for theability of viral protein to complement growth of the impaired virus,that is, the ability of the viral protein to provide interferonantagonist activity.

Thus, the following complementation assay was devised as an example ofan assay that could be used to test the ability of exogenous viralproteins to compensate for the delNS1 influenza A virus mutant'sinability to antagonize cellular interferon type I function.

6.1. EXPRESSION IN MDCK CELLS OF THE PR8 NS1 PROTEIN COMPLEMENTS GROWTHOF delNS1 VIRUS

The delNS1 virus grows poorly on MDCK cells compared with the wild-typePR8 influenza virus, a virus syngeneic with delNS1 virus except that itproduces the NS1 protein. It was therefore determined whether highefficiency transfection of MDCK cells with an NS1-expression plasmidwould complement growth of delNS1 virus. MDCK cells were transfectedusing Lipofectamin™2000 (GibcoBRL®) to introduce either an empty vector(pCAGGS) or an NS1 expression plasmid (pCAGGS-PR8 NS1 SAM) (Talon et al.2000 J.Virol. 74(17):7989-96). (“SAM” (spliceacceptor mutant) indicatesthat the splice acceptor within the NS1 ORF was mutated to preventexpression of an alternatively spliced message from the NS1 gene.)Sixteen hours post-transfection, the cells were infected with eitherwild-type PR8 or delNS1 virus at a multiplicity of infection (moi) of0.001. As a negative control, NS1-transfected cells were leftuninfected. Forty-eight hours post-transfection an HA assay wasperformed to determine viral titers (Table 2). TABLE 2 Transfection ofan NS1-expression plasmid complements growth on MDCK cells of delNS1virus. Plasmid Virus HA titer Empty vector delNS1 0 pCAGGS-NS1 SAMdelNS1 128 Empty vector PR8 32 pCAGGS-NS1 SAM PR8 128 pCAGGS-NS1 SAMnone 0

While delNS1 virus-infected, empty vector-transfected cells did notproduce a detectable HA titer, the delNS1-infected, NS1-transfectedcells yielded an HA titer equal to that achieved by infection withwild-type PR8. No HA titer was obtained when virus infection wasomitted. Thus, the restricted growth of delNS1 virus oninterferon-producing MDCK cells can be greatly enhanced by transfectionof an NS1 expression plasmid.

6.2. EXPRESSION IN MDCK CELLS OF THE INFLUENZA B VIRUS AND INFLUENZA CVIRUS NS1 PROTEINS ALSO COMPLEMENTS GROWTH OF delNS1 VIRUS

Based on the results in part 6.1, complementation of delNS1 growthshould also be possible following expression of other interferonantagonists. The influenza A, B and C NS1 proteins show little sequenceidentity to one another. However, the influenza B virus NS1 protein isable to bind RNA and to inhibit activation of PKR (Wang et al. 1999Virology 223(l):41-50). In addition, influenza B viruses encodingtruncated NS1 proteins have diminished ability to grow in interferonproducing embryonated chicken eggs. No data regarding the ability of theinfluenza C virus NS1 protein to bind RNA or inhibit PKR have beenreported. Furthermore, no data regarding the ability of influenza Cvirus NS1 protein to antagonize interferon responses have been reported.

Thus, the NS1 proteins encoded by the influenza B and C viruses weretested for delNS1 complementing activity. MDCK cells were transfected asdescribed above with an empty vector (pCAGGS), with the PR8 NS1expression plasmid (pCAGGS-PR8 NS1 SAM), a B/Yamagata/73 virus NS1expression plasmid (pCAGGS B NS1 SAM) or a C/Jhb/66 virus NS1 expressionplasmid (pCAGGS-C NS1 SAM). Sixteen hours post-transfection, the cellswere infected with delNS1 virus at an moi of 0.001. Tissue culturesupernatants were harvested forty eight hours post-infection. Plaqueassays were then performed to determine whether the A, B or C virus NS1proteins complemented growth of delNS1 virus (Table 3). The resultsindicate that both the influenza B virus and the influenza C virus NS1proteins, like the influenza A virus NS1 protein, can inhibitinterferon-mediated antiviral responses. TABLE 3 Complementation ofdelNS1 virus growth by influenza B virus NS1, influenza C virus NS1 andvaccinia virus E3L proteins. Titer Plasmid Virus (pfu/ml) Empty vectordelNS1   2 × 10² pCAGGS-PR8 NS1 SAM delNS1 2.5 × 10⁶ pCAGGS-B/Yam NS1SAM delNS1 3.7 × 10⁵ pCAGGS-C/Jhb NS1 SAM delNS1 2.8 × 10⁵ pCAGGS-E3LdelNS1   1 × 10⁵*Titer obtained by plaque assay 48 hours post-infection

6.3 EXPRESSION IN MDCK CELLS OF THE VACCINIA VIRUS E3L PROTEIN ALSOCOMPLEMENTS GROWTH OF delNS1 VIRUS

The vaccinia virus E3L protein is a dsRNA binding protein which can alsointeract directly with PKR (Chang et al. 1992 Proc. Natl. Acad. Sci. USA89(11):4825-9; Davies et al. 1993 J. Virol. 67(3):1688-92; Romano et al.1998 Mol. Cell. Biol. 18 (12):7304-16; Sharp et al. 1998 Virology250(2):302-15). E3L is able to inhibit PKR activity (Chang et al. 1992Proc. Natl. Acad. Sci. USA 89(11):4825-9), to inhibit OAS (Rivas et al.1998 Virology 243(2):406-14) and to protect vaccinia virus from theeffects of interferon (Beattie et al. 1995 J. Virol. 69(1)499-505; Shorset al. 1998 J. Interferon Cytokine Res. 18(9): 721-9). If the influenzaA, B and C virus NS1 proteins enhance growth of delNS1 virus on MDCKcells by inhibiting interferon responses, then the vaccinia virus E3Lprotein would also be predicted to complement delNS1 virus growth.Transfected E3L expression plasmid was indeed able to enhance growth ofdelNS1 virus on MDCK cells (Table 3).

It was determined that expression of another known inhibitor of the typeI IFN-induced antiviral response, HSV-1 ICP34.5, complements growth ofinfluenza delNS1 virus. Expression of the HSV-1-encoded PKR antagonistICP34.5 (Garcia-Sastre et al. 1998 Virology 252(2):324-30) clearlycomplemented growth of the influenza delNS1 virus (FIG. 5). This resultindicated that complementation of influenza delNS1 virus growth reflectsan anti-IFN function. This result also indicates that interferonantagonists encoded by viruses other than orthomyxoviruses can beidentified using the screening assays of the present invention.

7. EXAMPLE Expression in MDCK Cells of the Ebola Virus VP35 ProteinComplements Growth of delNS1 Virus

Ebola viruses are enveloped, negative-strand RNA viruses belonging tothe family Filoviridae. These viruses possess genomes of approximately19 kb and are known to encode eight proteins, the nucleoprotein (NP),VP35, VP40, glycoprotein (GP), soluble GP, VP30, VP24, and L(polymerase) proteins (Klenk et al. 1994 Encyclopedia of VirologyAcademic, New York vol 2: 827-31). Ebola virus infections frequentlyresult in severe hemorrhagic fever, and epidemics of the Ebola virus,Zaire subtype have resulted in mortality rates of greater than 80%(Klenk et al. 1994 Encyclopedia of Virology, Academic, New York, vol 2:827-31; Peters et al. 1999 Curr Top. Microb Immunol. 235:85-95). Thepathologic features and the immune responses characteristic of fatal andnonfatal human Ebola virus infections have begun to be characterized(Villinger et al. 1999 J. Infect. Dis. 179 Suppl. 1:S188-191; Yang etal. 1998 Science 279:1034-37). In order to determine if an ebola viralprotein exhibits interferon antagonist activity, the influenza delNS1virus complementation assay was used to screen for an ebola virusencoded interferon antagonist.

7.1. MATERIALS AND METHODS

Influenza delNS1 virus complementation assay. High-efficiency transienttransfection of MDCK cells was performed by using Lipofectamine 2000™(LF2000) (GIBCO/BRL). Four micrograms of the indicated expressionplasmid was adjusted to fifty microliters by Optimum I medium(GIBCO/BRL). Per transfection ten microliters of LF2000 was adjusted to0.25 ml with Optimum I medium and incubated in a five ml polystyrenesnap-cap tube at room temperature for five minutes. Each fiftymicroliter DNA sample was added to the 0.25 ml LF2000/Optimum I mixagitated gently, and incubated twenty minutes at room temperature. Aconfluent 80 cm² lask of MDCK cells was detached with trypsin. The cellswere brought to 12 ml wit hDMEM/10% fetal bovine serum (no antibiotics),pelleted at one thousand rpm for five minutes in a table top centrifugeand after aspiration of the supernatant resuspended in DMEM/10% Fetalbovine serum (no antibiotics) to a concentration of 4×10⁶ cells/ml. Aportion (0.25 ml) of the cell suspension was aliquoted in 35 mm tissueculture dishes. After twenty minutes incubation period one ml OfDMEM/10% FBS (no antibiotics) was added to each DNA/LF2000 mix and theDNA/LF2000 medium mixture was added to dishes containing the MDCK cells.After mixing the cells were maitained at 37° C. overnight. Sixteen totwenty hours posttransfection the cells were infected with 10³ plaqueforming units (PFU) of influenza delNS1 virus (multiplicity ofinfection=0.001) in a volume of 0.1 ml. After removal of the inoculum,the cells were maintained in 1.5 ml DMEM/0.3% bovine albumin/3micrograms/ml trypsin (trypsin 1:250 ;Difco)

7.2. RESULTS

To identify potential Ebola virus-encoded interferon antagonists,plasmids encoding Ebola virus proteins were screened for their abilityto complement growth of the delNS1 virus on MDCK cells (Table 4).Expression of the Ebola virus VP35 protein in MDCK cells was found tostimulate growth of the mutant influenza virus more than onethousand-fold. Therefore, the Ebola virus VP35 is likely to function asan interferon antagonist in Ebola virus infected cells. TABLE 3Complementation of delNS1 virus growth by Ebola virus proteins.Expressed protein pfu/ml Empty vector 10 NS1 1.2 × 10⁶ NP 10 VP35 1.9 ×10⁴ VP40 <10 GP <10 sGP 20 VP30 <10 VP24 <10

The Ebola Virus VP35 Protein Complements Growth of Influenza delNS1Protein. The influenza delNS1 virus complementation assay then was usedto screen for an Ebola virus-encoded IFN antagonist. An empty vector,the NS1-expression plasmid, or plasmids encoding individual Ebola virusproteins were transfected into MDCK cells. Twenty-four hoursposttransfection, the cells were infected with influenza delNS1 virus.Forty-eight hours postinfection, the supernatants were harvested andviral titers were determined by plaque assay (Table 4). The only Ebolavirus protein that enhanced influenza delNS1 virus growth was the VP35protein (Table 4). Time-course analysis clearly demonstrated theenhancement of influenza delNS1 virus growth by VP35 (FIG. 6).

Expression of the Ebola Virus VP35 Protein Blocks Induction of an ISREPromoter. To determine whether VP35 inhibits the dsRNA- andvirus-mediated activation of IFN-sensitive gene expression, cells weretransfected with an ISRE-driven CAT-reporter plasmid and aconstitutively expressed, simian virus 40 promoter-driven luciferasereporter plasmid. Additionally, the cells were transfected with emptyvector, NS1 expression plasmid, VP35 expression plasmid, or, as anadditional control, an Ebola virus NP expression plasmid. One day later,the cells were mock-treated, transfected with dsRNA, or infected witheither influenza delNS1 virus or with Sendai virus, strain Cantell (anattenuated strain known to induce large amounts of IFN). After anadditional twenty four hours, cell lysates were prepared and assayed forCAT activity and luciferase activity (FIG. 7A). Transfection of cellswith dsRNA or infection with either influenza delNS1 virus or Sendaivirus gave a strong induction of the IFN-sensitive promoter. When eitherNS1 or VP35 was present, expression from the IFN-responsive promoter wasalmost completely blocked. Levels of ISRE induction, normalized tolevels of luciferase activity, are shown in FIG. 7A. Expression of thecontrol luciferase reporter plasmid was not inhibited by expression ofeither NS1 or VP35. Expression of the Ebola virus NP, which did notcomplement growth of influenza delNS1 virus, did not inhibit activationof the ISRE promoter. Expression of the NS1, VP35, and NP proteins wasconfirmed by Western blotting (FIG. 7B). These results show that bothNS1 and VP35 can block type I IFN production and/or signaling inresponse to either dsRNA treatment or to viral infection.

Expression of the Ebola Virus VP35 Protein Blocks Activation of theINF-β Promoter. In wild-type influenza A virus-infected cells, the NS1protein blocks induction of type I IFN. This block is due, in largepart, to the ability of NS1 to prevent activation of IRF-3 and NF-B, twotranscription factors that play a critical role in stimulating thesynthesis of IFN-β. Synthesis of IFN-β, in turn, plays an important rolein the initiation of the type I IFN cascade (Marie et al. 1998 EMBO J.17:6660-69). The Ebola virus VP35, therefore, was tested for its abilityto block activation of the IFN-β promoter.

Empty vector, NS1 expression plasmid, or VP35 expression plasmid wascotransfected with a mouse IFN-β promoter-driven CAT reporter and asimian virus 40 promoter-driven luciferase reporter. When cellssubsequently were transfected with dsRNA, a strong induction of theIFN-β promoter was observed in empty vector-transfected cells, but thisinduction was blocked when either NS1 or VP35 was expressed (FIG. 8A).It also was determined whether VP35 could block activation of theendogenous human IFN-β promoter. Cells were transfected with emptyvector or VP35 expression plasmid and, twenty four hours later,mock-infected or infected with influenza delNS1 virus or with Sendaivirus. Ten or twenty hours postinfection, total cellular RNA wasisolated, and a Northern blot was performed to detect IFN-mRNA (FIG.8B). Expression of VP35 clearly blocked induction of the endogenousIFN-β promoter. Before infection with either virus, IFN-β mRNA wasundetectable. After infection, when the IFN-β mRNA levels werenormalized to β-actin mRNA levels, it was found that, in influenzadelNS1 virus-infected cells, the presence of VP35 reduced IFN-βinduction 8-fold at ten hours postinfection and 8.4-fold at twenty hoursposttransfection. In Sendai virus-infected cells, the presence of VP35reduced IFN-induction 6.1-fold at ten hours posttransfection and5.9-fold at twenty hours posttransfection.

The Ebola Virus VP35 Blocks INF Induction When Coexpressed with theEbola Virus NP. The VP35 protein is an essential component of the Ebolavirus RNA synthesis complex and likely associates with the viral NP(Muhlberger et al. 1999 J. Virol. 73:2333-42; Becker et al. 1998Virology 249:406-17). Therefore, it was determined whether Ebola virusVP35 retained its IFN-antagonizing properties when it was coexpressedwith the Ebola virus NP. An ISRE-reporter assay was performed in whichcells received either empty vector, VP35 alone, NP alone, or acombination of VP35 and NP. Twenty-four hours posttransfection, thecells were transfected with dsRNA or infected with Sendai virus. As seenpreviously, transfection with empty plasmid or with NP expressionplasmid did not block activation of the ISRE promoter, but expression ofVP35 did block its activation (FIG. 9). Further, coexpression of VP35and NP was able to block ISRE activation to the same extent asexpression of VP35 alone (FIG. 9). These data indicate that VP35, evenwhen coexpressed with the Ebola virus NP, can act as an IFN antagonist.

The Ebola virus VP35 protein inhibits type I IFN induction whencoexpressed with Ebola virus NP (FIG. 9). Fold induction of theIFN-inducible ISRE-driven reporter in the presence of empty vector,VP35, NP, or VP35 plus NP. 293 cells were transfected with a total of 4μg of expression plasmid, including 2 μg of a plasmid encoding anindividual protein and 2 μg of a second plasmid (either empty vector ora second expression plasmid) plus 0.3 μg each of the reporter plasmidspHISG-54-CAT and pGL2-Control. Twenty-four hours posttransfection, thecells were mock-treated or treated with the indicated IFN inducer.Twenty-four hours postinduction, CAT and luciferase assays wereperformed. The CAT activities were normalized to the correspondingluciferase activities to determine fold induction.

The production of an IFN antagonist contributes to the virulence ofEbola viruses. In humans, it appears that an appropriate cytokineresponse is related to the development of asymptomatic or nonfatal Ebolavirus infection. Thus, a viral factor that influences type I IFNproduction influences viral pathology.

8. EXAMPLE Complementation of Growth of Interferon-Sensitive Viruses byExpression of an Interferon Antagonist, the influenza A Virus NS1Protein

In the example below an influenza A NS1 (PR8) was shown to enhance thegrowth of a virus with impaired interferon antagonist activity.

8.1. INFLUENZA C VIRUS GROWTH IS RESTRICTED IN EMBRYONATED CHICKEN EGGSTHAT PRODUCE AN INTERFERON RESPONSE

Influenza C virus was tested for its ability to grow in 7-day old versus11-day old embryonated chicken eggs. Young embryos, such as 7-day oldembryos, produce little interferon in response to viral infection whileolder embryos, such as 11-day old embryos, produce higher levels ofinterferon in response to viral infection (Sekellick et al. 1990 Invitro Cell Biol. 26:997-1003). Replication of influenza C/Jhb/66 viruswas found to be significantly more efficient in the younger eggs (Table5). These data strongly suggest that growth of influenza C virus isrestricted by interferon. TABLE 5 Growth of influenza C/Jhb/66 virus in7- and 11-day old embryonated chicken eggs.* Age of embryo (days) HAtiter 7 512 11 4*Eggs were inoculated with 500 pfu of virus and incubated for 3 days at33° C.

8.2. EXPRESSION IN MDCK CELLS OF THE PR8 NS1 PROTEIN ENHANCES GROWTH OFINFLUENZA C VIRUS

Given the sensitivity of influenza C virus to interferon, the ability ofa potent interferon antagonist (the influenza A virus NS1 protein) toenhance influenza C virus growth on MDCK cells was tested. Theexperiment was performed similarly to that described for delNS1 virusexcept that transfected cells were infected with influenza C/Jhb/66virus (moi.=0.001) instead of delNS1 virus. The results are shown inTable 6. TABLE 6 Expression of the influenza A virus NS1 proteincomplements growth of influenza C virus on MDCK cells. Plasmid HA titerEmpty plasmid 2 pCAGGS-PR8 NS1 32

Thus expression of a potent viral interferon antagonist can enhancegrowth of viruses which are sensitive to the effects of interferon. Theexpression of the viral interferon antagonist may be used for theisolation, growth and analysis of interferon-sensitive viruses.

9. EXAMPLE Complementation of Growth of an Interferon-Sensitive Virus byan Interferon Antagonist Derived from a Paramyxovirus

In the example below, respiratory syncytial virus (RSV) NS2 was shown tobe an interferon antagonist using the screening assays described herein.The expression of RSV NS2 was shown to support the growth of anattenuated non-RSV virus with impaired interferon antagonist activity.

9.1 EXPRESSION IN MDCK CELLS OF THE RESPIRATORY SYNCITIAL VIRUS (RSV)NS2 PROTEIN COMPLEMENTS GROWTH OF delNS1 VIRUS

Human RSV is the leading cause of severe viral respiratory infections inchildren. Although it has been reported that the NS1 and NS2 proteins ofbovine RSV have interferon antagonistic properties the human RSV geneproducts responsible for antagonizing interferon are unknown. Toidentify potential human RSV-encoded interferon antagonists, plasmidsencoding human RSV proteins were screened for their ability tocomplement growth of the delNS1 virus on MDCK cells(Table 7). Expressionof the human RSV NS2 protein in MDCK cells was found to stimulate growthof the mutant influenza virus. Therefore, the human RSV NS2 proteinfunctions as an interferon antagonist. TABLE 7 RSV N2 complements growthof del Ns1 Vims Plasmid Virus HA titer Empty vector delNS1 0pCDNA3-PR8NS1 delNS1 128 SAM pcDNA3-hRSV NS2 delNS1 16Titer obtained by hemagglutination assay 48 hours post infection

10. EXAMPLE Co-Transfection of the Influenza A Virus NS1 ProteinEnhances Expression from Co-Transfected Expression Plasmids

The following example demonstrates the ability of an interferonantagonist to enhance translation of mRNAs.

The influenza A virus NS1 protein has been reported to enhancetranslation of mRNAs (de la Luna et al. 1995 J.Virol. 67(4):2427-33;Enami et al. 1994 J. Virol. 68(3):1432-37). This ability is likelyrelated to its ability to inhibit activation of the interferon-induceddsRNA-activated protein kinase, PKR (Hatada et al. 1999 J. Virol.73(3):2425-33). However, it is not clear whether NS1 inhibits PKR bysequestering dsRNA (Lu et al. 1999 Virology 214(1):222-28), byinteracting directly with PKR (Tan et al. 1998 J. Interferon CytokineRes. 18(9):757-66) or by a combination of the two mechanisms. Theability to enhance translation is a property characteristic of severalviral-encoded PKR inhibitors, including adenovirus VA RNA₁ (Svensson etal. 1985 EMBO J.4(4):957-64) the vaccinia virus E3L protein (Davies etal. 1993 J. Virol. 67(3):1688-92), and perhaps the hepatitis C virusNS5A protein (Gale et al. 1997 Virology 230(2):217-27). These proteinsalso appear to confer interferon-resistance to the viruses (Beattie etal., 1995 J. Virol 69(1):499-505; Kitajewski et al. 1986 Cell45(2):195-200).

Therefore, the ability of the PR8 NS1 expression plasmid to enhanceexpression from a co-transfected reporter plasmid was tested. 293T cellswere transfected with a total of 6 μg DNA. The 6 μg consisted of 4 μgpGL2-Control (Promega Corp.) (an SV40-promoter-driven, constitutivelyexpressed luciferase reporter plasmid), 1 μg pEGFP-c1 (ClonetechLaboratories) (a CMV-promoter-driven green fluorescence protein (GFP)expression plasmid) and a combination of PCAGGS and pCAGGS-PR8 NS1 SAMtotaling leg. Transfections were performed containing 0, 1, 0.2 and 0.04μg NS1 expression plasmid. Forty eight hours post-transfection, thecells were observed for GFP expression to confirm that dishes weretransfected at comparable levels, and luciferase assays were performed.NS1-expression plasmid gave a 19.8-fold maximal stimulation ofluciferase expression, and the enhancement was dose-dependent (FIG. 5).

Thus, interferon antagonists identified using the screening assaysdescribed herein have utility in enhancing translation of mRNAs in invitro and in vivo applications.

The present invention is not to be limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

All publications cited herein are incorporated by reference in theirentirety.

1. A screening method for identifying a viral protein with aninterferon-antagonizing function comprising: (a) contacting a cell whichexpresses NS1 protein, with a virus containing a mutation that resultsin a decrease in activity of a viral polypeptide; (b) identifying amutant whose growth in the cell is enhanced by the presence of NS1protein; and (c) identifying the viral polypeptide as having aninterferon antagonizing function.
 2. The screening method of claim 1wherein the virus is a paramyxovirus.
 3. The screening method of claim 1wherein the virus is a morbillivirus.
 4. The screening method of claim 1wherein the virus is a pneumovius.
 5. The screening method of claim 1wherein the virus is a rhabdovirus.
 6. A screening method foridentifying a potential antiviral agent comprising: (a) contacting acell that expresses (i) a reporter gene operatively linked to aninterferon responsive promoter element and (ii) an interferonantagonist, with a test agent, following stimulation of a cellularinterferon response; (b) monitoring a level of reporter gene product;(c) identifying the test agent as a potential antiviral agent when itspresence results in an increase in reporter gene product.
 7. Thescreening method of claim 6, wherein the reporter gene product is greenfluorescence protein (GFP).
 8. The screening method of claim 6, whereinthe interferon antagonist is NS1 protein.
 9. The screening method ofclaim 6, wherein the interferon antagonist is E3L protein.
 10. Thescreening method of claim 6, wherein the interferon antagonist is VP35protein.